Albumin-fused kunitz domain peptides

ABSTRACT

The invention relates to proteins comprising serine protease inhibiting peptides, such as Kunitz domain peptides (including, but not limited to, fragments and variants thereof) fused to albumin, or fragments or variants thereof. These fusion proteins are herein collectively referred to as “albumin fusion proteins of the invention.” These fusion proteins exhibit extended shelf-life and/or extended or therapeutic activity in solution. The invention encompasses, therapeutic albumin fusion proteins, compositions, pharmaceutical compositions, formulations and kits. The invention also encompasses nucleic acid molecules encoding the albumin fusion proteins of the invention, as well as vectors containing these nucleic acids, host cells transformed with these nucleic acids and vectors, and methods of making the albumin fusion proteins of the invention using these nucleic acids, vectors, and/or host cells. The invention also relates to compositions and methods for inhibiting neutrophil elastase, kallikrein, and plasmin. The invention further relates to compositions and methods for treating cystic fibrosis and cancer.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/355,547, filed Feb. 7, 2002. The disclosure of that applicationis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the fields of Kunitz domain peptides andalbumin fusion proteins. More specifically, the invention relates toKunitz domain peptides and albumin fusion proteins for treating,preventing, or ameliorating a disease or disorder.

BACKGROUND OF THE INVENTION

A Kunitz domain is a folding domain of approximately 51-64 residueswhich forms a central anti-parallel beta sheet and a short C-terminalhelix (see e.g., U.S. Pat. No. 6,087,473, which is hereby incorporatedby reference in its entirety). This characteristic domain comprises sixcysteine residues that form three disulfide bonds, resulting in adouble-loop structure. Between the N-terminal region and the first betastrand resides the active inhibitory binding loop. This binding loop isdisulfide bonded through the P2 C₁₄ residue to the hairpin loop formedbetween the last two beta strands. Isolated Kunitz domains from avariety of proteinase inhibitors have been shown to have inhibitoryactivity (e.g., Petersen et al., Eur. J. Biochem. 125:310-316, 1996;Wagner et al., Biochem. Biophys. Res. Comm. 186:1138-1145, 1992; Denniset al., J. Biol. Chem. 270:25411-25417, 1995).

Linked Kunitz domains also have been shown to have inhibitory activity,as discussed, for example, in U.S. Pat. No. 6,087,473. Proteinaseinhibitors comprising one or more Kunitz domains include tissue factorpathway inhibitor (TFPI), tissue factor pathway inhibitor 2 (TFPI-2),amyloid β-protein precursor (AβPP), aprotinin, and placental bikunin.TFPI, an extrinsic pathway inhibitor and a natural anticoagulant,contains three tandemly linked Kunitz inhibitor domains. Theamino-terminal Kunitz domain inhibits factor VIIa, plasmin, andcathepsin G; the second domain inhibits factor Xa, trypsin, andchymotrypsin; and the third domain has no known activity (Petersen etal., ibid.).

The inhibitory activity of Kunitz domain peptides towards serineproteases has been demonstrated in several previous studies. Thefollowing subsections discuss studies of the inhibition of serineproteases, such as plasma kallikrein, plasmin, and neutrophil elastaseby Kunitz Domain peptides.

Plasma Kallikrein Inhibitors

Kallikreins are serine proteases found in both tissues and plasma [see,for example, U.S. Pat. No. 6,333,402 to Markland, which is herebyincorporated by reference in its entirety]. Plasma kallikrein isinvolved in contact-activated (intrinsic pathway) coagulation,fibrinolysis, hypotension, and inflammation [See Bhoola, K. D., C. D.Figueroa, and K. Worthy, Pharmacological Reviews (1992) 44(1)1-80].These effects of kallikrein are mediated through the activities of threedistinct physiological substrates:

-   -   i) Factor XII (coagulation),    -   ii) Pro-urokinase/plasminogen (fibrinolysis), and    -   iii) Kininogens (hypotension and inflammation).

Kallikrein cleavage of kininogens results in the production of kinins,small highly potent bioactive peptides. The kinins act through cellsurface receptors, designated BK-1 and BK-2, present on a variety ofcell types including endothelia, epithelia, smooth muscle, neural,glandular and hematopoietic. Intracellular heterotrimeric G-proteinslink the kinin receptors to second messenger pathways including nitricoxide, adenyl cyclase, phospholipase A₂ and phospholipase C. Among thesignificant physiological activities of kinins are: (i) increasedvascular permeability; (ii) vasodilation; (iii) bronchospasm; and (iv)pain induction. Thus, kinins mediate the life-threatening vascular shockand edema associated with bacteremia (sepsis) or trauma, the edema andairway hyperreactivity of asthma, and both inflammatory and neurogenicpain associated with tissue injury. The consequences of inappropriateplasma kallikrein activity and resultant kinin production aredramatically illustrated in patients with hereditary angioedema (HAE).HAE is due to a genetic deficiency of C1-inhibitor, the principalendogenous inhibitor of plasma kallikrein. Symptoms of HAE include edemaof the skin, subcutaneous tissues and gastrointestinal tract, andabdominal pain and vomiting. Nearly one-third of HAE patients die bysuffocation due to edema of the larynx and upper respiratory tract.Kallikrein is secreted as a zymogen (prekallikrein) that circulates asan inactive molecule until activated by a proteolytic event. [Genebankentry P03952 shows Human Plasma Prekallikrein.]

An important inhibitor of plasma kallikrein (pKA) in vivo is the C1inhibitor; (see Schmaier, et al. in “Contact Activation and ItsAbnormalities”, Chapter 2 in Hemostasis and Thrombosis, Colman, R W, JHirsh, V J Marder, and E W Salzman, Editors, Second Edition, 1987, J. B.Lippincott Company, Philadelphia, Pa., pp. 27-28). Cl is a serpin andforms an irreversible or nearly irreversible complex with pKA. Althoughbovine pancreatic trypsin inhibitor (also known as BPTI, aprotinin, orTrasylol™) was initially thought to be a strong pKA inhibitor withK_(i)=320 pM [Auerswald, E.-A., D. Hoerlein, G. Reinhardt, W. Schroder,and E. Schnabel, Bio. Chem. Hoppe-Seyler, (1988), 369(Supplement):27-35], a more recent report [Berndt, et al., Biochemistry,32:4564-70, 1993] indicates that its K₁ for plasma Kallikrein is 30 nM(i.e., 30,000 pM). The G36S mutant had a K_(i) of over 500 nM.

Markland et al. [U.S. Pat. Nos. 6,333,402; 5,994,125; 6,057,287; and5,795,865; each reference hereby incorporated by reference in itsentirety] claim a number of derivatives having high affinity andspecificity in inhibiting human plasma kallikrein. One of these proteinsis being tested in human patients who have HAE. Although earlyindications are that the compound is safe and effective, the duration ofeffect is shorter than desired.

Plasmin Inhibitors

Plasmin is a serine protease derived from plasminogen. The catalyticdomain of plasmin (or “CatDom”) cuts peptide bonds, particularly afterarginine residues and to a lesser extent after lysines and is highlyhomologous to trypsin, chymotrypsin, kallikrein, and many other serineproteases. Most of the specificity of plasmin derives from the kringles'binding of fibrin (Lucas et al., J Biological Chem (1983)258(7)4249-56.; Varadi & Patthy, Biochemistry (1983) 22:2440-2446.; andVaradi & Patthy, Biochemistry (1984) 23:2108-2112.). On activation, thebond between ARG₅₆₁-Val₅₆₂ is cut, allowing the newly free aminoterminus to form a salt bridge. The kringles remain, nevertheless,attached to the CatDom through two disulfides (Colman, R W, J Hirsh, V JMarder, and E W Salzman, Editors, Hemostasis and Thrombosis, SecondEdition, 1987, J. B. Lippincott Company, Philadelphia, Pa., Bobbins,1987, supra.

The agent mainly responsible for fibrinolysis is plasmin the activatedform of plasminogen. Many substances can activate plasminogen, includingactivated Hageman factor, streptokinase, urokinase (uPA), tissue-typeplasminogen activator (tPA), and plasma kallikrein (pKA). pKA is both anactivator of the zymogen form of urokinase and a direct plasminogenactivator.

Plasmin is undetectable in normal circulating blood, but plasminogen,the zymogen, is present at about 3 μM. An additional, unmeasured amountof plasminogen is bound to fibrin and other components of theextracellular matrix and cell surfaces. Normal blood contains thephysiological inhibitor of plasmin, α₂-plasmin inhibitor (α₂-PI), atabout 2 μM. Plasmin and α2-PI form a 1:1 complex. Matrix or cellbound-plasmin is relatively inaccessible to inhibition by α₂-PI. Thus,activation of plasmin can exceed the neutralizing capacity of α₂-PIcausing a profibrinolytic state.

Plasmin, once formed:

-   -   i) degrades fibrin clots, sometimes prematurely;    -   ii) digests fibrinogen (the building material of clots)        impairing hemostasis by causing formation of friable, easily        lysed clots from the degradation products, and inhibition of        platelet adhesion/aggregation by the fibrinogen degradation        products;    -   iii) interacts directly with platelets to cleave glycoproteins        Ib and IIb/IIIa preventing adhesion to injured endothelium in        areas of high shear blood flow and impairing the aggregation        response needed for platelet plug formation (Adelman et al.,        Blood (1986) 68(6)1280-1284.);    -   iv) proteolytically inactivates enzymes in the extrinsic        coagulation pathway further promoting a prolytic state. Robbins        (Robbins, Chapter 21 of Hemostasis and Thrombosis, Colman, R.        W., J. Hirsh, V. J. Marder, and E. W. Salzman, Editors, Second        Edition, 1987, J. B. Lippincott Company, Philadelphia, Pa.)        reviewed the plasminogen-plasmin system in detail. This        publication (i.e., Colman, R. W., J Hirsh, V. J. Marder,        and E. W. Salzman, Editors, Hemostasis and Thrombosis, Second        Edition, 1987, J. B. Lippincott Company, Philadelphia, Pa.) is        hereby incorporated by reference.        Fibrinolysis and Fibrinogenolysis

Inappropriate fibrinolysis and fibrinogenolysis leading to excessivebleeding is a frequent complication of surgical procedures that requireextracorporeal circulation, such as cardiopulmonary bypass, and is alsoencountered in thrombolytic therapy and organ transplantation,particularly liver. Other clinical conditions characterized by highincidence of bleeding diathesis include liver cirrhosis, amyloidosis,acute promyelocytic leukemia, and solid tumors. Restoration ofhemostasis requires infusion of plasma and/or plasma products, whichrisks immunological reaction and exposure to pathogens, e.g. hepatitisvirus and HIV.

Very high blood loss can resist resolution even with massive infusion.When judged life-threatening, the hemorrhage is treated withantifibrinolytics such as c-amino caproic acid (See Hoover et al.,Biochemistry (1993) 32:1093643) (EACA), tranexarnic acid, or aprotinin(Neuhaus et al., Lancet (1989) 2(8668)924-5). EACA and tranexamic acidonly prevent plasmin from binding fibrin by binding the kringles, thusleaving plasmin as a free protease in plasma. BPTI is a direct inhibitorof plasmin and is the most effective of these agents. Due to thepotential for thrombotic complications, renal toxicity and, in the caseof BPTI, immunogenicity, these agents are used with caution and usuallyreserved as a “last resort” (Putterman, Acta Chir Scand (1989)155(6-7)367). All three of the antifibrinolytic agents lack targetspecificity and affinity and interact with tissues and organs throughuncharacterized metabolic pathways. The large doses required due to lowaffinity, side effects due to lack of specificity and potential forimmune reaction and organ/tissue toxicity augment against use of theseantifibrinolytics prophylactically to prevent bleeding or as a routinepostoperative therapy to avoid or reduce transfusion therapy. Thus,there is a need for a safe antifibrinolytic. The essential attributes ofsuch an agent are:

-   -   i) Neutralization of relevant target fibrinolytic enzyme(s);    -   ii) High affinity binding to target enzymes to minimize dose;    -   iii) High specificity for target, to reduce side effects; and    -   iv) High degree of similarity to human protein to minimize        potential immunogenicity and organ/tissue toxicity.

All of the fibrinolytic enzymes that are candidate targets forinhibition by an efficacious antifibrinolytic are chymotrypin-homologousserine proteases.

Excessive Bleeding

Excessive bleeding can result from deficient coagulation activity,elevated fibrinolytic activity, or a combination of the two conditions.In most bleeding diatheses one must control the activity of plasmin. Theclinically beneficial effect of BPTI in reducing blood loss is thoughtto result from its inhibition of plasmin (K_(i) ˜0.3 nM) or of plasmakallikrein (K_(i)˜100 nM) or both enzymes.

Gardell [Toxicol. Pathol. (1993) 21(2)190-8] has reviewed currently-usedthrombolytics, and has stated that, although thrombolytic agents (e.g.tPA) do open blood vessels, excessive bleeding is a serious safetyissue. Although tPA and streptokinase have short plasma half lives, theplasmin they activate remains in the system for a long time and, asstated, the system is potentially deficient in plasmin inhibitors. Thus,excessive activation of plasminogen can lead to a dangerous inability toclot and injurious or fatal hemorrhage. A potent, highly specificplasmin inhibitor would be useful in such cases.

BPTI is a potent plasmin inhibitor. However, it has been found that itis sufficiently antigenic that second uses require skin testing.Furthermore, the doses of BPTI required to control bleeding are quitehigh and the mechanism of action is not clear. Some say that BPTI actson plasmin while others say that it acts by inhibiting plasmakallikrein. Fraedrich et al. [Thorac Cardiovasc Surg (1989) 37(2)89-91]report that doses of about 840 mg of BPTI to 80 open-heart surgerypatients reduced blood loss by almost half and the mean amounttransfused was decreased by 74%. Miles Inc. has recently introducedTrasylol™ in the U.S. for reduction of bleeding in surgery [see Milesproduct brochure on Trasylol™, which is hereby incorporated byreference]. Lohmann and Marshal [Refract Corneal Surg (1993) 9(4)300-2]suggest that plasmin inhibitors may be useful in controlling bleeding insurgery of the eye. Sheridan et al. [Dis Colon Rectum (1989) 32(6)505-8]reports that BPTI may be useful in limiting bleeding in colonic surgery.

A plasmin inhibitor that is approximately as potent as BPTI or morepotent but that is almost identical to a human protein domain offerssimilar therapeutic potential but poses less potential for antigenicity.

Angiogenesis:

Plasmin is the key enzyme in angiogenesis. O'Reilly et al. [Cell (1994)79:315-328] reports that a 38 kDa fragment of plasmin (lacking thecatalytic domain) is a potent inhibitor of metastasis, indicating thatinhibition of plasmin could be useful in blocking metastasis of tumors[Fidler & Ellis, Cell (1994) 79:185-188; See also Ellis et al., Ann NYAcad Sci (1992) 667:13-31; O'Reilly et al., Fidler & Ellis, and Ellis etal. are hereby incorporated by reference].

Neutrophil Elastase Inhibition

Cystic Fibrosis is a hereditary, autosomal recessive disorder affectingpulmonary, gastrointestinal, and reproductive systems. With a prevalenceof 80,000 worldwide, the incidence of CF is estimated at 1 in 3500[Cystic Fibrosis Foundation, Patient Registry 1998 Annual Data Report,Bethesda, Md., September 1999]. The genetic defect in CF was describedin 1989 as the loss of a single phenylalanine at position 508 (ΔF508),resulting in a faulty cystic fibrosis transmembrane conductanceregulator protein (CFTR) which inhibits the reabsorption of Cl⁻ (andhence Na⁺ and water) [Rommens, J. M., et al., “Identification of thecystic fibrosis gene: chromosome walking and jumping,” Science 245:1059,1989; Riordan, J. R., et al., “Identification of the cystic fibrosisgene: cloning and complementary DNA,” Science 245:1066, 1989; Kerem, B.,et al., “Identification of the cystic fibrosis gene: genetic analysis,Science 245:1073, 1989]. Mutations other than ΔF508 have been found inCFTR and may cause CF. Desiccated mucus then plugs many of thepassageways in the respiratory, gastrointestinal, and reproductivesystems.

More than 75% of the mortality from CF is due to respiratorycomplications [Cystic Fibrosis Foundation, Patient Registry 1998 AnnualData Report, Bethesda, Md., September 1999]. Although disease of thepancreas, liver, and intestine is present in CF individuals beforebirth, the CF lung is normal at birth and until the onset of infectionand inflammation. Then, defective Cl⁻ reabsorption in the CF lung leadsto desiccated airway secretions by drawing sodium out of the airways,with water following passively. Desiccated secretions may then interferewith mucociliary clearance by trapping bacteria in an environment wellsuited to colonization with distinctive microbial pathogens [Reynolds,H. Y., et al., “Mucoid Pseudomonas aeruginosa: a sign of cystic fibrosisin young adults with chronic pulmonary disease,” J.A.M.A. 236:2190,1976]. The ensuing lung infection and inflammation recruits andactivates neutrophils which release neutrophil elastase (NE). Theneutrophil-dominated inflammation on the respiratory epithelial surfaceresults in a chronic epithelial burden of neutrophil elastase.Endogenous antiprotease is rapidly overwhelmed by an excess of NE in theCF lung. In addition, NE stimulates the production of pro-inflammatorymediators and cleaves complement receptors and IgG, thereby cripplinghost defense mechanisms preventing further bacterial colonization [Tosi,M. F., et al., “Neutrophil elastase cleaves C3bi on opsonizedPseudomonas as well as CR1 on neutrophils to create a functionallyimportant opsonin receptor mismatch,” J. Clin. Invest. 86:300, 1990].The infection thereby becomes persistent, and the massive ongoinginflammation and excessive levels of NE destroy the airway epithelium,leading to bronchiectasis, and the progressive loss of pulmonaryfunction and death.

One therapeutic approach in patients with CF is the eradication of CFpathogens by systemic antimicrobials such as tobramycin and ciprofloxin.While these specific antimicrobial agents have been shown to beeffective in clearing infection and improving pulmonary function,antibiotic resistance to tobramycin and ciprofloxin is reported in 7.5%and 9.6% of CF patients respectively [Cystic Fibrosis Foundation,Patient Registry 1998 Annual Data Report, Bethesda, Md., September1999]. As the use of these antimicrobials for CF increases in patientsof whom 60% are infected with P. aeruginosa and 41% with S. aureus, drugresistance selection pressure has increased.

Pulmonary function also has been a therapeutic target in patients withCF. Pulmozyme® (domase alfa), a recombinant human deoxyribonucleasewhich reduces mucus viscoelasticity by hydrolyzing DNA in sputum, hasbeen shown in clinical studies to increase FEV₁ and FVC after 8 days oftreatment. This change last for six months, and is accompanied by areduction in the use of intravenous antibiotics [Fuchs, H. L., et al.,“Effect of aerosolized recombinant human Dnase on exacerbations ofrespiratory symptoms and on pulmonary function in patients with cysticfibrosis,” N. Engl. J. Med., 331:637-642, 1994].

Another therapeutic approach is to use a protease inhibitor to ablatethe direct effect of NE on elastase degradation and its sequelae.Neutralization of excess NE can restore normal homeostatic balance whichprotects the extracellular lung matrix. Normalized antiprotease activityin the lung preserves elastin, reduces mucus viscosity through reductionof the neutrophil response, and preserves of pulmonary function, thusreducing mortality in CF. In addition, the restoration ofcomplement-mediated phagocytosis can enable the immune system to clearbacterial pathogens, resulting in reduction of the incidence, duration,and severity of pulmonary infection. For example, in a rat model of CF,after seven days of treatment with alpha₁ antitrypsin reduced bacterialcounts to 0.2±0.4, compared to 85±21 in the placebo group [Cantin, A.and Woods, D, “Aerosolized Prolastin Suppresses Bacterial Proliferationin a Model of Chronic Pseudomonas aeruginosa Lung Infection” Am J RespirCrit Care Med 160:1130-1136,1999].

SUMMARY OF THE INVENTION

The invention relates to proteins comprising Kunitz domain peptidesfused to albumin. These fusion proteins are herein collectively referredto as “albumin fusion proteins of the invention.” These fusion proteinsof the invention exhibit extended in vivo half-life and/or extended ortherapeutic activity in solution.

The invention encompasses therapeutic albumin fusion proteins,compositions, pharmaceutical compositions, formulations and kits. Theinvention also encompasses nucleic acid molecules encoding the albuminfusion proteins of the invention, as well as vectors containing thesenucleic acids, host cells transformed with these nucleic acids andvectors, and methods of making the albumin fusion proteins of theinvention using these nucleic acids, vectors, and/or host cells.

An object of the invention is to provide an albumin fusion proteincomprising a Kunitz domain peptide or a fragment or variant thereof, andalbumin, or a fragment or variant thereof. Suitable Kunitz domainpeptides for use in such albumin fusion proteins include DX-890, DX-88,DX-1000, and DPI-14. The Kunitz domain peptide portion optionally may beseparated from the albumin portion by a linker. Another object of theinvention is to provide compositions and methods involving albuminfusion proteins for inhibiting serine proteases, non-limiting examplesof which include plasma kallikrein, plasmin and neutrophil elastase.

Another aspect of the invention is to provide an albumin fusion proteincomprising at least two Kunitz domain peptides or fragments or variantsthereof, wherein at least one of the Kunitz domain peptide or fragmentor variant has a functional activity, such as inhibiting plasmin,kallikrein, or human neutrophil elastase.

Yet another aspect of this invention is to provide an albumin fusionprotein comprising a Kunitz domain peptide, or a fragment or variantthereof, and albumin, or a fragment or variant thereof, wherein thealbumin has an albumin activity that prolongs the in vivo half-life of aKunitz domain peptide, such as DX-890, DX-88, DX-1000, and DPI-14, or afragment or variant thereof, compared to the in vivo half-life of theKunitz domain peptide or a fragment or variant thereof in an unfusedstate.

Yet another aspect of this invention is to provide an albumin fusionprotein comprising a Kunitz domain peptide, or a fragment or variantthereof, and albumin, or a fragment of variant thereof, wherein thealbumin fusion protein of the invention has increased solubility atphysiological pH.

One aspect of the invention is to provide an albumin fusion proteincomprising a Kunitz domain peptide, or fragment or variant thereof, andalbumin, or fragment or variant thereof, wherein the Kunitz domainpeptide, or fragment or variant thereof, is fused to the N-terminus ofalbumin or to the N-terminus of the fragment or variant of albumin.Alternatively, this invention also provides an albumin fusion proteincomprising a Kunitz domain peptide, or fragment or variant thereof, andalbumin, or fragment or variant thereof, wherein the Kunitz domainpeptide, or fragment or variant thereof, is fused to the C-terminus ofalbumin or to the C-terminus of the fragment or variant of albumin.

This invention provides a composition comprising an albumin fusionprotein and a pharmaceutically acceptable carrier. Another object of theinvention is to provide a method of treating a patient with cysticfibrosis, a cystic fibrosis-related disease or disorder, or a disease ordisorder that can be modulated by a Kunitz domain peptide comprisingDX-890 and/or DPI-14. The method comprises the step of administering aneffective amount of the albumin fusion protein comprising a Kunitzdomain peptide that comprises DX-890 and/or DPI-14, or fragment orvariant thereof, and albumin, or fragment or variant thereof.

Another object of this invention is to provide a method of treating apatient with hereditary angioedema, a hereditary angioedema-relateddisease or disorder, or a disease that is modulated by a Kunitz domainpeptide such as DX-88. The method comprises the step of administering aneffective amount of the albumin fusion protein, wherein the albuminfusion protein comprises a Kunitz domain peptide comprising DX-88, orfragment or variant thereof, and albumin, or fragment or variantthereof.

An object of this invention is to provide a method of treating a patientwith cancer, a cancer-related disease, bleeding, or disease that ismodulated by a Kunitz domain peptide such as DX-1000. The methodcomprises the step of administering an effective amount of the albuminfusion protein, wherein the albumin fusion protein comprises a Kunitzdomain peptide comprising DX-1000, or fragment or variant thereof, andalbumin, or fragment or variant thereof.

Another object of the invention is to provide a nucleic acid moleculecomprising a polynucleotide sequence encoding an albumin fusion protein,as well as a vector that comprises such a nucleic acid molecule.

The invention also provides a method for manufacturing a albumin fusionprotein, wherein the method comprises:

-   -   (a) providing a nucleic acid comprising a nucleotide sequence        encoding the albumin fusion protein expressible in an organism;    -   (b) expressing the nucleic acid in the organism to form an        albumin fusion protein; and    -   (c) purifying the albumin fusion protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: K_(i) measurements of DX-890 and the DX-890-HSA fusion.

FIG. 2: Plasma clearance curves for ¹²⁵I-DX-890 (left) and¹²⁵I-DX-890-HSA fusion (right).

FIG. 3: ¹²⁵I-DX890 in normal mouse plasma on SE-HPLC (Superose-12).

FIG. 4: SE-HPLC(Superose-12) Profiles of ¹²⁵I-HAS-DX890 in normal mouseplasma.

FIG. 5: Plasma Clearance of ¹²⁵I Labeled DX-890 and HSA-DX-890 inRabbits

FIG. 6: SEC Analysis of Rabbit Plasma Samples

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to albumin-fused Kunitz domain peptides.The present invention also relates to bifunctional (or multifunctional)fusion proteins in which albumin is coupled to two (or more) Kunitzdomain peptides, optionally different Kunitz domain peptides. Suchbifunctional (or multifunctional) fusion proteins having differentKunitz domain peptides are expected to have an improved drug resistanceprofile as compared to an albumin fusion protein comprising only onetype of Kunitz domain peptide. Some conditions may require inhibition oftwo or more proteases and fusion of multiple Kunitz domains allows onecompound to be used for inhibition of the two or more proteases.Alternatively, one can fuse two or more Kunitz domains, each directed tothe same protease so that the inhibitor activity per gram is increased.A useful form of an inhibitor having two Kunitz domains is K₁::SA::K₂,where K₁ and K₂ are the Kunitz domains and SA is serum albumin or asubstantial portion thereof. Such bifunctional (or multifunctional)fusion proteins may also exhibit synergistic effects, as compared to analbumin fusion protein comprising only one type of Kunitz domainpeptide. Furthermore, chemical entities may be covalently attached tothe fusion proteins of the invention to enhance a biological activity orto modulate a biological activity.

The albumin fusion proteins of the present invention are expected toprolong the half-life of the Kunitz domain peptide in vivo. The in vitroor in vivo half-life of said albumin-fused peptide is extended 2-fold,or 5-fold, or more, over the half-life of the peptide lacking the linkedalbumin. Furthermore, due at least in part to the increased half-life ofthe peptide, the albumin fusion proteins of the present invention areexpected to reduce the frequency of the dosing schedule of thetherapeutic peptide. The dosing schedule frequency is reduced by atleast one-quarter or by at least one-half, as compared to the frequencyof the dosing schedule of the therapeutic peptide lacking the linkedalbumin.

The albumin fusion proteins of the present invention prolong the shelflife of the peptide, and/or stabilize the peptide and/or its activity insolution (or in a pharmaceutical composition) in vitro and/or in vivo.These albumin fusion proteins, which may be therapeutic agents, areexpected to reduce the need to formulate protein solutions with largeexcesses of carrier proteins (such as albumin, unfused) to prevent lossof proteins due to factors such as nonspecific binding.

The present invention also encompasses nucleic acid molecules encodingthe albumin fusion proteins as well as vectors containing these nucleicacids, host cells transformed with these nucleic acids vectors, andmethods of making the albumin fusion proteins of the invention usingthese nucleic acids, vectors, and/or host cells. The present inventionfurther includes transgenic organisms modified to contain the nucleicacid molecules of the invention, optionally modified to express thealbumin fusion proteins encoded by the nucleic acid molecules.

Albumin

The terms, human serum albumin (HSA) and human albumin (HA) are usedinterchangeably herein. The terms, “albumin” and “serum albumin” arebroader, and encompass human serum albumin (and fragments and variantsthereof) as well as albumin from other species (and fragments andvariants thereof).

As used herein, “albumin” refers collectively to albumin protein oramino acid sequence, or an albumin fragment or variant, having one ormore functional activities (e.g., biological activities) of albumin. Inparticular, “albumin” refers to human albumin or fragments thereof (seeEP 201 239, EP 322 094 WO 97/24445, WO95/23857) especially the matureform of human albumin as shown in SEQ ID NO:18 herein and in Table 1 andSEQ ID NO:18 of U.S. Provisional Application Ser. No. 60/355,547 and WO01/79480 or albumin from other vertebrates or fragments thereof, oranalogs or variants of these molecules or fragments thereof.

The human serum albumin protein used in the albumin fusion proteins ofthe invention contains one or both of the following sets of pointmutations with reference to SEQ ID NO:18: Leu-407 to Ala, Leu-408 toVal, Val-409 to Ala, and Arg410 to Ala; or Arg-410 to Ala, Lys-413 toGln, and Lys-414 to Gln (see, e.g., International Publication No.WO95/23857, hereby incorporated in its entirety by reference herein). Insome embodiments, albumin fusion proteins of the invention that containone or both of above-described sets of point mutations have improvedstability/resistance to yeast Yap3p proteolytic cleavage, allowingincreased production of recombinant albumin fusion proteins expressed inyeast host cells.

As used herein, a portion of albumin sufficient to prolong or extend thein vivo half-life, therapeutic activity, or shelf-life of theTherapeutic protein refers to a portion of albumin sufficient in lengthor structure to stabilize, prolong or extend the in vivo half-life,therapeutic activity or shelf life of the Therapeutic protein portion ofthe albumin fusion protein compared to the in vivo half-life,therapeutic activity, or shelf-life of the Therapeutic protein in thenon-fusion state. The albumin portion of the albumin fusion proteins maycomprise the full length of the HA sequence as described above, or mayinclude one or more fragments thereof that are capable of stabilizing orprolonging the therapeutic activity. Such fragments may be of 10 or moreamino acids in length or may include about 15, 20, 25, 30, 50, or morecontiguous amino acids from the HA sequence or may include part or allof specific domains of HA.

The albumin portion of the albumin fusion proteins of the invention maybe a variant of normal HA. The Therapeutic protein portion of thealbumin fusion proteins of the invention may also be variants of theTherapeutic proteins as described herein. The term “variants” includesinsertions, deletions and substitutions, either conservative ornon-conservative, where such changes do not substantially alter one ormore of the oncotic, useful ligand-binding and non-immunogenicproperties of albumin, or the active site, or active domain whichconfers the therapeutic activities of the Therapeutic proteins.

In particular, the albumin fusion proteins of the invention may includenaturally occurring polymorphic variants of human albumin and fragmentsof human albumin, for example those fragments disclosed in EP 322 094(namely HA (Pn), where n is 369 to 419). The albumin may be derived fromany vertebrate, especially any mammal, for example human, cow, sheep, orpig. Non-mammalian albumins include, but are not limited to, hen andsalmon. The albumin portion of the albumin fusion protein may be from adifferent animal than the Therapeutic protein portion.

Generally speaking, an HA fragment or variant will be at least 100 aminoacids long, for example, at least 150 amino acids long. The HA variantmay consist of or alternatively comprise at least one whole domain ofHA, for example domains 1 (amino acids 1-194 of SEQ ID NO:18), 2 (aminoacids 195-387 of SEQ ID NO:18), 3 (amino acids 388-585 of SEQ ID NO:18),1+2 (1-387 of SEQ ID NO:18), 2+3 (195-585 of SEQ ID NO:18) or 1+3 (aminoacids 1-194 of SEQ ID NO:18+amino acids 388-585 of SEQ ID NO:18). Eachdomain is itself made up of two homologous subdomains namely 1-105,120-194, 195-291, 316-387, 388-491 and 512-585, with flexibleinter-subdomain linker regions comprising residues Lys106 to Glu119,Glu292 to Val315 and Glu492 to Ala511.

The albumin portion of an albumin fusion protein of the invention maycomprise at least one subdomain or domain of HA or conservativemodifications thereof. If the fusion is based on subdomains, some or allof the adjacent linker may optionally be used to link to the Therapeuticprotein moiety.

Albumin Fusion Proteins

The present invention relates generally to albumin fusion proteins andmethods of treating, preventing, or ameliorating diseases or disorders.As used herein, “albumin fusion protein” refers to a protein formed bythe fusion of at least one molecule of albumin (or a fragment or variantthereof) to at least one molecule of a Therapeutic protein (or fragmentor variant thereof). An albumin fusion protein of the inventioncomprises at least a fragment or variant of a Therapeutic protein and atleast a fragment or variant of human serum albumin, which are associatedwith one another, such as by genetic fusion (i.e., the albumin fusionprotein is generated by translation of a nucleic acid in which apolynucleotide encoding all or a portion of a Therapeutic protein isjoined in-frame with a polynucleotide encoding all or a portion ofalbumin) to one another. The Therapeutic protein and albumin protein,once part of the albumin fusion protein, may be referred to as a“portion”, “region”, or “moiety” of the albumin fusion protein.

In one embodiment, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a Therapeutic protein and asemm albumin protein. In other embodiments, the invention provides analbumin fusion protein comprising, or alternatively consisting of, abiologically active and/or therapeutically active fragment of aTherapeutic protein and a serum albumin protein. In other embodiments,the invention provides an albumin fusion protein comprising, oralternatively consisting of, a biologically active and/ortherapeutically active variant of a Therapeutic protein and a serumalbumin protein. In some embodiments, the serum albumin proteincomponent of the albumin fusion protein is the mature portion of serumalbumin.

In further embodiments, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a Therapeutic protein, and abiologically active and/or therapeutically active fragment of serumalbumin. In further embodiments, the invention provides an albuminfusion protein comprising, or alternatively consisting of, a Therapeuticprotein and a biologically active and/or therapeutically active variantof serum albumin. In certain embodiments, the Therapeutic proteinportion of the albumin fusion protein is the mature portion of theTherapeutic protein.

In further embodiments, the invention provides an albumin fusion proteincomprising, or alternatively consisting of, a biologically active and/ortherapeutically active fragment or variant of a Therapeutic protein anda biologically active and/or therapeutically active fragment or variantof serum albumin. In some embodiments, the invention provides an albuminfusion protein comprising, or alternatively consisting of, the matureportion of a Therapeutic protein and the mature portion of serumalbumin.

The albumin fusion protein comprises HA as the N-terminal portion, and aTherapeutic protein as the C-terminal portion. Alternatively, an albuminfusion protein comprising HA as the C-terminal portion, and aTherapeutic protein as the N-terminal portion may also be used.

In other embodiments, the albumin fusion protein has a Therapeuticprotein fused to both the N-terminus and the C-terminus of albumin. Inone embodiment, the Therapeutic proteins fused at the N- and C-terminiare the same Therapeutic proteins. In another embodiment, theTherapeutic proteins fused at the N- and C-termini are differentTherapeutic proteins. In yet another embodiment, the Therapeuticproteins fused at the N- and C-termini are different Therapeuticproteins which may be used to treat or prevent the same disease,disorder, or condition. In some embodiments, the Therapeutic proteinsfused at the N- and C-termini are different Therapeutic proteins whichmay be used to treat or prevent diseases or disorders which are known inthe art to commonly occur in patients simultaneously.

In addition to albumin fusion protein in which the albumin portion isfused N-terminal and/or C-terminal of the Therapeutic protein portion,albumin fusion proteins of the invention may also be produced byinserting the Therapeutic protein or peptide of interest into aninternal region of HA. For instance, within the protein sequence of theHA molecule a number of loops or turns exist between the end andbeginning of α-helices, which are stabilized by disulphide bonds. Theloops, as determined from the crystal structure of HA (PDB identifiers1AO6, 1BJ5, 1BKE, 1BM0, 1E7E to 1E7I and 1UOR) for the most part extendaway from the body of the molecule. These loops are useful for theinsertion, or internal fusion, of therapeutically active peptides,particularly those requiring a secondary structure to be functional, orTherapeutic proteins, to essentially generate an albumin molecule withspecific biological activity.

Loops in human albumin structure into which peptides or polypeptides maybe inserted to generate albumin fusion proteins of the inventioninclude: Val54-Asn61, Thr76-Asp89, Ala92-Glu100, Gln170-Ala176,His247-Glu252, Glu266-Glu277, Glu280-His288, Ala362-Glu368,Lys439-Pro447, Val462-Lys475, Thr478-Pro486, and Lys560-Thr566. In otherembodiments, peptides or polypeptides are inserted into the Val54-Asn61,Gln170-Ala176, and/or Lys560-Thr566 loops of mature human albumin(Table 1) (SEQ ID NO:18).

The Therapeutic protein to be inserted may be derived from any source,including phage display and synthetic peptide libraries screened forspecific biological activity or from the active portions of a moleculewith the desired function. Additionally, random peptide librariescomprising Kunitz domain peptides that are candidates for use as aTherapeutic protein may be generated within particular loops or byinsertions of such randomized peptides into particular loops of the HAmolecule and in which many (e.g. 5×10⁹) combinations of amino acids arerepresented.

Such library(s) could be generated on HA or domain fragments of HA byone of the following methods:

-   -   (a) randomized mutation of amino acids within-one or more        peptide loops of HA or HA domain fragments. Either one, more        than one or all the residues within a loop could be mutated in        this manner;    -   (b) replacement of, or insertion into one or more loops of HA or        HA domain fragments (i.e., internal fusion) of a randomized        peptide(s) of length X_(n) (where X is an amino acid and n is        the number of residues;    -   (c) N-, C- or N- and C-terminal peptide/protein fusions in        addition to (a) and/or (b).

The HA or HA domain fragment may also be made multifunctional bygrafting the peptides derived from different screens of different loopsagainst different targets into the same HA or HA domain fragment.

Non-limiting examples of peptides inserted into a loop of human serumalbumin are DX-890 (an inhibitor of human neutrophil elastase), DPI-14(an inhibitor of human neutrophil elastase), DX-88 peptide (an inhibitorof human plasma kallikrein, Table 2), and DX-1000 (an inhibitor of humanplasmin, Table 2) or peptide fragments or peptide variants thereof. Moreparticularly, the invention encompasses albumin fusion proteins whichcomprise peptide fragments or peptide variants at least 7 at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 20, at least 25, at least 30, at least 35, orat least 40 amino acids in length inserted into a loop of human serumalbumin. The invention also encompasses albumin fusion proteins whichcomprise peptide fragments or peptide variants at least 7 at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 20, at least 25, at least 30, at least 35, orat least 40 amino acids fused to the N-terminus of human serum albumin.The invention also encompasses albumin fusion proteins which comprisepeptide fragments or peptide variants at least 7 at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 20, at least 25, at least 30, at least 35, or atleast 40 amino acids fused to the C-terminus of human serum albumin.

Generally, the albumin fusion proteins of the invention may have oneHA-derived region and one Therapeutic protein-derived region. Multipleregions of each protein, however, may be used to make an albumin fusionprotein of the invention. Similarly, more than one Therapeutic proteinmay be used to make an albumin fusion protein of the invention. Forinstance, a Therapeutic protein may be fused to both the N- andC-terminal ends of the HA. In such a configuration, the Therapeuticprotein portions may be the same or different Therapeutic proteinmolecules. The structure of bifunctional albumin fusion proteins may berepresented as: X-HA-Y or Y-HA-X or X-Y-HA or HA-X-Y or HA-X-Y-HA orHA-Y-X-HA or HA-X-X-HA or HA-Y-Y-HA or HA-X-HA-Y or X-HA-Y-HA ormultiple combinations or inserting X and/or Y within the HA sequence atany location.

Additional embodiments that involve a therapeutic protein “X”, such as aKunitz domain, and a therapeutic peptide “Y” involve separating HA intoparts 1 and 2. The fusion proteins of the invention could have theforms: X-HA(part1)-Y-HA(part2) and HA(part1)-Y-HA(part2)-X. Additionalembodiments involve two therapeutic protein domains “X” and “Z” and atherapeutic peptide “Y” leading to fusion proteins of the forms:X-HA(part1)-Y-HA(part2)-Z and Z-HA(part1)-Y-HA(part2)-X.

Bi- or multi-functional albumin fusion proteins may be prepared invarious ratios depending on function, half-life, etc.

Bi- or multi-functional albumin fusion proteins may also be prepared totarget the Therapeutic protein portion of a fusion to a target organ orcell type via protein or peptide at the opposite terminus of HA.

As an alternative to the fusion of known therapeutic molecules, thepeptides could be obtained by screening libraries constructed as fusionsto the N-, C- or N- and C-termini of HA, or domain fragment of HA, oftypically 6, 8, 12, 20 or 25 or X_(n) (where X is an amino acid (aa) andn equals the number of residues) randomized amino acids, and in whichall possible combinations of amino acids were allowed. A particularadvantage of this approach is that the peptides may be selected in situon the HA molecule and the properties of the peptide would therefore beas selected for rather than, potentially, modified as might be the casefor a peptide derived by any other method then being attached to HA.Such selection is not needed for attachment of well-folded domains, suchas Kunitz domains, at the ends of HA. Selection in-situ is likely to beimportant for peptides that have no disulfides or a single disulfideloop.

Additionally, the albumin fusion proteins of the invention may include alinker peptide between the fused portions to provide greater physicalseparation between the moieties and thus maximize the accessibility ofthe Therapeutic protein portion, for instance, for binding to itscognate receptor. The linker peptide may consist of amino acids suchthat it is flexible or more rigid.

Therefore, as described above, the albumin fusion proteins of theinvention may have the following formula R2-R1; R1-R2; R2-R1-R2;R2-L-R-L-R2; R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, wherein R1 is at leastone Therapeutic protein, peptide or polypeptide sequence (includingfragments or variants thereof), and not necessarily the same Therapeuticprotein, L is a linker and R2 is a serum albumin sequence (includingfragments or variants thereof). Exemplary linkers include (GGGGS)^(N)(SEQ ID NO:______ or (GGGS)^(N) (SEQ ID NO:______) or (GGS)^(N), whereinN is an integer greater than or equal to 1 and wherein G representsglycine and S represents serine.

In certain embodiments, albumin fusion proteins of the inventioncomprising a Therapeutic protein have extended shelf life or in vivohalf-life or therapeutic activity compared to the shelf life or in vivohalf-life or therapeutic activity of the same Therapeutic protein whennot fused to albumin. Shelf-life typically refers to the time periodover which the therapeutic activity of a Therapeutic protein in solutionor in some other storage formulation, is stable without undue loss oftherapeutic activity. Many of the Therapeutic proteins are highly labilein their unfused state. As described below, the typical shelf-life ofthese Therapeutic proteins is markedly prolonged upon incorporation intothe albumin fusion protein of the invention.

Albumin fusion proteins of the invention with “prolonged” or “extended”shelf-life exhibit greater therapeutic activity relative to a standardthat has been subjected to the same storage and handling conditions. Thestandard may be the unfused full-length Therapeutic protein. When theTherapeutic protein portion of the albumin fusion protein is an analog,a variant, or is otherwise altered or does not include the completesequence for that protein, the prolongation of therapeutic activity mayalternatively be compared to the unfused equivalent of that analog,variant, altered peptide or incomplete sequence. As an example, analbumin fusion protein of the invention may retain greater than about100% of the therapeutic activity, or greater than about 105%, 110%,120%, 130%, 150% or 200% of the therapeutic activity of a standard whensubjected to the same storage and handling conditions as the standardwhen compared at a given time point. However, it is noted that thetherapeutic activity depends on the Therapeutic protein's stability, andmay be below 100%.

Shelf-life may also be assessed in terms of therapeutic activityremaining after storage, normalized to therapeutic activity when storagebegan. Albumin fusion proteins of the invention with prolonged orextended shelf-life as exhibited by prolonged or extended therapeuticactivity may retain greater than about 50% of the therapeutic activity,about 60%, 70%, 80%, or 90% or more of the therapeutic activity of theequivalent unfused Therapeutic protein when subjected to the sameconditions.

Albumin fusion proteins of the invention exhibit greater solubilityrelative to the non-fused Therapeutic protein standard that has beensubjected to the same storage and handling conditions.

Therapeutic Proteins

As stated above, an albumin fusion protein of the invention comprises atleast a fragment or variant of a Therapeutic protein and at least afragment or variant of human serum albumin, which are associated withone another by genetic fusion.

As used herein, “Therapeutic protein” refers to a Kunitz domain peptide,non-limiting examples of which include DX-890, DPI-14, DX-88 or DX-1000,or fragments or variants thereof, having one or more therapeutic and/orbiological activities. A Kunitz domain is a folding domain ofapproximately 51-64 residues which forms a central anti-parallel betasheet and a short C-terminal helix. This characteristic domain comprisessix cysteine residues that form three disulfide bonds, resulting in adouble-loop structure. Between the N-terminal region and the first betastrand resides the active inhibitory binding loop. This binding loop isdisulfide bonded through the P2 C₁₄ residue to the hairpin loop formedbetween the last two beta strands.

A Kunitz domain is a polypeptide of from about 51 AAs to about 64 AAs ofthe form: (SEQ ID NO: _(——))X₁X₂X₃X₄C₅X₆X₇X₈X₉X_(9a)X₁₀X₁₁X₁₂X₁₃C₁₄X₁₅X₁₆X₁₇X₁₈X₁₉₋X₂₀X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X_(26a)X_(26b) X_(26c)X₂₇X₂₈X₂₉C₃₀X₃₁X₃₂-X₃₃X₃₄X₃₅X₃₆X₃₇C₃₈X₃₉X₄₀X₄₁X_(42X) _(42a)X_(42b)X₄₃X₄₄X₄₅-X₄₆X₄₇X₄₈X₄₉C₅₀X₅₁X₅₂X₅₃X₅₄C₅₅X₅₆X₅₇X₅₈

Disulfides are formed between C₅ and C₅₅, C₁₄ and C₃₈, and C₃₀ and C₅₁.The C₁₄-C₃₈ disulfide is always seen in natural Kunitz domains, but maybe removed in artificial Kunitz domains. If C₁₄ is changed to anotheramino-acid type, then C₃₈ is also changed to a non-cysteine and viceversa. Any polypeptide may be fused to the amino terminus. X₁-X₄ maycomprise zero to four amino acids. X₆-X₁₃ may comprise 8 or 9 aminoacids. If X_(9a) is absent, then X₁₂ is Gly. Each Of X_(26a), X_(26b),and X_(26c) may be absent; that is, X₁₅-X₃₀ may comprise 16, 17, 18, or19 amino acids. X₃₃ is Phe or Tyr. X₃₉-X₅₀ may comprise 12, 13, 14, or15 amino acids; that is, each of X_(42A), X_(42b), and X_(42c) may beabsent. X₄₅ is Phe or Tyr. X₅₆-X₅₈ may comprise zero to three aminoacids. Additional cysteines may occur at positions 50, 53, 54 or 58. Anypolypeptide may be fused to the carboxy terminus. Table 3 shows theamino-acid sequences of 21 known human Kunitz domains. TABLE 3 Aminoacid sequences of 21 known human Kunitz domains Domain Protein AminoAcid Sequence Accession single A4 (amyloid VREVCSEQAETGPCRAMISRWYFDVTEGKSP:A4_HUMAN precursor CAPFFYGGCGGNRNNFDTEEYCMAVCGSA A#P05067 PTN) SEQ IDNO:_(——) single embl loCus KQDVCEMPKETGPCLAYFLHWWYDKKDNT (CAB37635;HS461P17 CSMFVYGGCQGNNNNFQSKANCLNTCKNK g4467797 “CAB37” SEQ ID NO:_(——)single Amyloid-like VKAVCSQEAMTGPCRAVMPRWYFDLSKGK Loc:1703344;S41082 PTN2 CVRFIYGGCGGNRNNFESEDYCMAVCKAM g1082207 & SEQ ID NO:_(——) g1703344 &g477608 K1 ITI KEDSCQLGYSAGPCMGMTSRYFYNGTSMA SP:HC_HUMAN,CETFQYGGCMGNGNNFVTEKECLQTCRTV A#P02760 (HI-8e) - SEQ ID NO:_(——)gi|223133 K2 ITI TVAACNLPIVRGPCRAFIQLWAFDAVKGK SP:HC HUMAN,CVLFPYGGCQGNGNKFYSEKECREYCGVP A#P02760 (HI-8e) - SEQ ID NO:_(——)gi|223133 K1 TFPI-1 = MHSFCAFKADDGPCKAIMKRFFFNIFTRQ SP:HC_HUMAN, LACICEEFIYGGCEGNQNRFESLEECKKMCTRD A#P10646 gim|14667 N SEQ ID NO:_(——)(corrected 2000.05.14) K2 TFPI-1 KPDFCFLEEDPGICRGYITRYFYNNQTKQSP:LACI_HUMAN, CERFKYGGCLGNMNNFETLEECKNICEDG A#P10646 gim|14667 SEQ IDNO:_(——) K3 TFPI-1 GPSWCLTPADRGLCRANENRFYYNSVIGK SP:LACI_HUMAN,CRPFKYSGCGGNENNFTSKQECLRACKKG A#P10646 gim|14667 SEQ ID NO:_(——) K1TFPI-2 NAEICLLPLDYGPCRALLLRYYYDRYTQS Specher &al. PNASCRQFLYGGCEGNANNFYTWEACDDACWRI 91:3353-3357 (1994) SEQ ID NO:_(——) K2TFPI-2 VPKVCRLQVVDDQCEGSTEKYFFNLSSMT Specher &al, PNASCEKFFSGGCHRNRNRFPDEATCMGFCAPK 91:3353ff (1994) SEQ ID NO:_(——) K3 TFPI-2IPSFCYSPKDEGLCSANVTRYYFNPRYRT Specher &al, PNASCDAFTYTGCGGNDNNFVSREDCKRACAKA 91:3353ff (1994) SEQ ID NO:_(——) K1Hepatocyte TEDYCLASNKVGRCRGSFPRWYYDPTEQI LOCUS 2924601 GF activatorCKSFVYGGCLGNKNNYLREEECILACRGV inhib type 1 SEQ ID NO:_(——) K2 HepatocyteDKGHCVDLPDTGLCKESIPRWYYNPFSEH LOCUS 2924601 GF activatorCARFTYGGCYGNKNNFEEEQQCLESCRGI inhib type 1 SEQ ID NO:_(——) K1 hepatocyteIHDFCLVSKVVGRCRASMPRWWYNVTDGS LOG 2924620 GF activatorCQLFVYGGCDGNSNNYLTKEECLKKCATV inhib. type 2 SEQ ID NO:_(——) K2hepatocyte YEEYCTANAVTGPCRASFPRWYFDVERNS LOG 2924620 GF activatorCNNFIYGGCRGNKNSYRSEEACMLRCFRQ inhib. type2 SEQ ID NO:_(——) Single PRFTVAACNLPVIRGPCRAFIQLWAFDAVKGK gi|223132 CVLFPYGGCQGNGNKFYSEKECREYCGVPName: 0511271A SEQ ID NO:_(——) Single 1-HKI B9LPNVCAFPMEKGPCQTYMTRWFFNFETGE gi|579567 domainCELFAYGGCGGNSNNFLRKEKCEKFCKFT WO93/14123-A; SEQ ID NO:_(——) g542925Single Collagen ∀1 SDDPCSLPLDEGSCTAYTLRWYHRAVTEA NCBI: gi|543915 (VII)CHPFVYGGCGGNANRFGTREACERRCPPR SEQ ID NO:_(——) Single collagen alphaEDDPCSLPLDEGSCTAYTLRWYHRAVTGS g627406- A54849 1(VII)TEACHPFVYGGCGGNANRFGTREACERRC GI:627406 PPR SEQ ID NO:_(——) Singlecollagen ∀3 ETDICKLPKDEGTCRDFILKWYYDPNTKS NGBI Seq ID: 512802CARFWYGGCGGNENKFGSQKECEKVCAPV WO93/14119-A. SEQ ID NO:_(——) 2193976(Xray) single Chromosome FQEPCMLPVRHGNCNHEAQRWHFDFKNYR CAB37634 20ptnCTPFKYRGCEGNANNFLNEDACRTACMLI PID g702435O “Chrome20” SEQ ID NO:_(——)

Any of the domains in Table 1 could be engineered to have a specificbiological effect (such as inhibiting a particular protease) and befused to HA. Thus an albumin fusion protein of the invention may containat least a fragment or variant of a Therapeutic protein. Variantsinclude mutants, analogs, and mimetics, as well as homologs, includingthe endogenous or naturally occurring correlates.

By a polypeptide displaying a “therapeutic activity” or a protein thatis “therapeutically active” is meant a polypeptide that possesses one ormore known biological and/or therapeutic activities associated with aTherapeutic protein such as one or more of the Therapeutic proteinsdescribed herein or otherwise known in the art. As a non-limitingexample, a “Therapeutic protein” is a protein that is useful to treat,prevent or ameliorate a disease, condition or disorder.

As used herein, “therapeutic activity” or “activity” may refer to anactivity whose effect is consistent with a desirable therapeutic outcomein humans, or to desired effects in non-human mammals or in otherspecies or organisms. Therapeutic activity may be measured in vivo or invitro. For example, a desirable effect may be assayed in cell culture.Such in vitro or cell culture assays are commonly available for manyTherapeutic proteins as described in the art.

Examples of useful assays include, but are not limited to, thosedescribed in references and publications of Table 4, specificallyincorporated by reference herein, and those described in the Examplesherein. The activity exhibited by the fusion proteins of the inventionmay be measured, for example, by easily performed in vitro assays, suchas those described herein. Using these assays, such parameters as therelative biological and/or therapeutic activity that the fusion proteinsexhibit as compared to the Therapeutic protein (or fragment or variantthereof) when it is not fused to albumin can be determined.

Therapeutic proteins corresponding to a Therapeutic protein portion ofan albumin fusion protein of the invention may be modified by theattachment of one or more oligosaccharide groups. The modification,referred to as glycosylation, can dramatically affect the physicalproperties of proteins and can be important in protein stability,secretion, and localization. Such modifications are described in detailin U.S. Provisional Application Ser. No. 60/355,547 and WO 01/79480,which are incorporated herein by reference.

Therapeutic proteins corresponding to a Therapeutic protein portion ofan albumin fusion protein of the invention, as well as analogs andvariants thereof, may be modified so that glycosylation at one or moresites is altered as a result of manipulation(s) of their nucleic acidsequence, by the host cell in which they are expressed, or due to otherconditions of their expression. For example, glycosylation isomers maybe produced by abolishing or introducing glycosylation sites, e.g., bysubstitution or deletion of amino acid residues, such as substitution ofglutamine for asparagine, or unglycosylated recombinant proteins may beproduced by expressing the proteins in host cells that will notglycosylate them, e.g. in E. coli or glycosylation-deficient yeast.Examples of these approaches are described in more detail in U.S.Provisional Application Ser. No. 60/355,547 and WO 01/79480, which areincorporated by reference, and are known in the art.

Table 4 provides a non-exhaustive list of Therapeutic proteins thatcorrespond to a Therapeutic protein portion of an albumin fusion proteinof the invention. The “Therapeutic Protein X” column disclosesTherapeutic protein molecules followed by parentheses containingscientific and brand names that comprise, or alternatively consist of,that Therapeutic protein molecule or a fragment or variant thereof.“Therapeutic protein X” as used herein may refer either to an individualTherapeutic protein molecule (as defined by the amino acid sequenceobtainable from the CAS and Genbank accession numbers), or to the entiregroup of Therapeutic proteins associated with a given Therapeuticprotein molecule disclosed in this column. The information associatedwith each of these entries are each incorporated by reference in theirentireties, particularly with respect to the amino acid sequencesdescribed therein. The “PCT/Patent Reference” column provides U.S.Patent numbers, or PCT International Publication Numbers correspondingto patents and/or published patent applications that describe theTherapeutic protein molecule. Each of the patents and/or publishedpatent applications cited in the “PCT/Patent Reference” column areherein incorporated by reference in their entireties. In particular, theamino acid sequences of the specified polypeptide set forth in thesequence listing of each cited “PCT/Patent Reference”, the variants ofthese amino acid sequences (mutations, fragments, etc.) set forth, forexample, in the detailed description of each cited “PCT/PatentReference”, the therapeutic indications set forth, for example, in thedetailed description of each cited “PCT/Patent Reference”, and theactivity assays for the specified polypeptide set forth in the detaileddescription, and more particularly, the examples of each cited“PCT/Patent Reference” are incorporated herein by reference. The“Biological activity” column describes Biological activities associatedwith the Therapeutic protein molecule. Each of the references cited inthe “Relevant Publications” column are herein incorporated by referencein their entireties, particularly with respect to the description of therespective activity assay described in the reference (see Methodssection, for example) for assaying the corresponding biologicalactivity. The “Preferred Indication Y” column describes disease,disorders, and/or conditions that may be treated, prevented, diagnosed,or ameliorated by Therapeutic protein X or an albumin fusion protein ofthe invention comprising a Therapeutic protein X portion. TABLE 4 A Listof Selected Therapeutic Proteins Therapeutic PCT/Patent BiologicalRelevant Protein X Reference Activity Publications Preferred IndicationY DX-890, U.S. Pat. No. Inhibition of Rusckowski et al. Emphysema,Cystic DPI14 5,663,143, human neutrophil (2000) J. Nuclear fibrosisCOPD, SEQ ID elastase, K_(j)˜5 pM. Medicine 41: 363-74 Bronchitis,Pulmonary NO:20 = Hypertension, Acute DX-890 respiratory distresssyndrome, Interstitial lung disease, Asthma, Smoke intoxication,Bronchopulmonary dysplasia, Pneumonia, Thermal Injury, Lung transplantrejection. DX-88 U.S. Pat. Nos. Inhibition of Markland et al. HAE6,333,402; human plasma Biochemistry 5,994,125; kallikrein 35(24):8058-67, 6,057,287; 1996. and Ley et al. (1996) 5,795,865 Mol Divers2(1-2) 119-24. DX-1000 U.S. Pat. Nos. Inhibits human Markland et al.Bleeding, cancer. 6,010,880; plasmin Biochemistry 6,071,723; 35(24):8045-57, and 1996. 6,103,499 Ley et al. (1996) Mol Divers 2(1-2) 119-24.

In various embodiments, the albumin fusion proteins of the invention arecapable of a therapeutic activity and/or biologic activity correspondingto the therapeutic activity and/or biologic activity of the Therapeuticprotein corresponding to the Therapeutic protein portion of the albuminfusion protein listed in the corresponding row of Table 4. (See, e.g.,the “Biological Activity” and “Therapeutic Protein X” columns of Table4.) In other embodiments, the therapeutically active protein portions ofthe albumin fusion proteins of the invention are fragments or variantsof the reference sequence and are capable of the therapeutic activityand/or biologic activity of the corresponding Therapeutic proteindisclosed in “Biological Activity” column of Table 4.

Polypeptide and Polynucleotide Fragments and Variants

Fragments

The present invention is further directed to fragments of theTherapeutic proteins described in Table 4, albumin proteins, and/oralbumin fusion proteins of the invention.

Even if deletion of one or more amino acids from the N-terminus of aprotein results in modification or loss of one or more biologicalfunctions of the Therapeutic protein, albumin protein, and/or albuminfusion protein, other Therapeutic activities and/or functionalactivities (e.g., biological activities, ability to multimerize, abilityto bind a ligand) may still be retained. For example, the ability ofpolypeptides with N-terminal deletions to induce and/or bind toantibodies which recognize the complete or mature forms of thepolypeptides generally will be retained when less than the majority ofthe residues of the complete polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a mutein with a large numberof deleted N-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixamino acid residues may often evoke an immune response.

Accordingly, fragments of a Therapeutic protein corresponding to aTherapeutic protein portion of an albumin fusion protein of theinvention, include the full length protein as well as polypeptideshaving one or more residues deleted from the amino terminus of the aminoacid sequence of the reference polypeptide (e.g., a Therapeutic proteinas disclosed in Table 4). Polynucleotides encoding these polypeptidesare also encompassed by the invention.

In addition, fragments of serum albumin polypeptides corresponding to analbumin protein portion of an albumin fusion protein of the invention,include the full length protein as well as polypeptides having one ormore residues deleted from the amino terminus of the amino acid sequenceof the reference polypeptide (i.e., serum albumin). Polynucleotidesencoding these polypeptides are also encompassed by the invention.

Moreover, fragments of albumin fusion proteins of the invention includethe full-length albumin fusion protein as well as polypeptides havingone or more residues deleted from the amino terminus of the albuminfusion protein. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the N-terminus or C-terminus of a reference polypeptide (e.g., aTherapeutic protein and/or serum albumin protein) results inmodification or loss of one or more biological functions of the protein,other functional activities (e.g., biological activities, ability tomultimerize, ability to bind a ligand) and/or Therapeutic activities maystill be retained. For example the ability of polypeptides withC-terminal deletions to induce and/or bind to antibodies which recognizethe complete or mature forms of the polypeptide generally will beretained when less than the majority of the residues of the complete ormature polypeptide are removed from the C-terminus. Whether a particularpolypeptide lacking the N-terminal and/or C-terminal residues of areference polypeptide retains Therapeutic activity can readily bedetermined by routine methods described herein and/or otherwise known inthe art.

The present invention further provides polypeptides having one or moreresidues deleted from the carboxy terminus of the amino acid sequence ofa Therapeutic protein corresponding to a Therapeutic protein portion ofan albumin fusion protein of the invention (e.g., a Therapeutic proteinreferred to in Table 4). Polynucleotides encoding these polypeptides arealso encompassed by the invention.

In addition, the present invention provides polypeptides having one ormore residues deleted from the carboxy terminus of the amino acidsequence of an albumin protein corresponding to an albumin proteinportion of an albumin fusion protein of the invention (e.g., serumalbumin). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Moreover, the present invention provides polypeptides having one or moreresidues deleted from the carboxy terminus of an albumin fusion proteinof the invention. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

In addition, any of the above described N- or C-terminal deletions canbe combined to produce a N- and C-terminal deleted reference polypeptide(e.g., a Therapeutic protein referred to in Table 4, or serum albumin(e.g., SEQ ID NO:18, Table 1), or an albumin fusion protein of theinvention). The invention also provides polypeptides having one or moreamino acids deleted from both the amino and the carboxyl termini.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

The present application is also directed to proteins containingpolypeptides at least 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a reference polypeptide sequence (e.g., a Therapeuticprotein, serum albumin protein or an albumin fusion protein of theinvention) set forth herein, or fragments thereof. In some embodiments,the application is directed to proteins comprising polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to referencepolypeptides having the amino acid sequence of N- and C-terminaldeletions as described above. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Other polypeptide fragments of the invention are fragments comprising,or alternatively, consisting of, an amino acid sequence that displays aTherapeutic activity and/or functional activity (e.g. biologicalactivity) of the polypeptide sequence of the Therapeutic protein orserum albumin protein of which the amino acid sequence is a fragment.

Other polypeptide fragments are biologically active fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the polypeptide of thepresent invention. The biological activity of the fragments may includean improved desired activity, or a decreased undesirable activity.

Variants

“Variant” refers to a polynucleotide or nucleic acid differing from areference nucleic acid or polypeptide, but retaining essentialproperties thereof. Generally, variants are overall closely similar,and, in many regions, identical to the reference nucleic acid orpolypeptide.

As used herein, “variant”, refers to a Therapeutic protein portion of analbumin fusion protein of the invention, albumin portion of an albuminfusion protein of the invention, or albumin fusion protein differing insequence from a Therapeutic protein (e.g., see “Therapeutic Protein X”column of Table 4), albumin protein, and/or albumin fusion protein ofthe invention, respectively, but retaining at least one functionaland/or therapeutic property thereof (e.g., a therapeutic activity and/orbiological activity as disclosed in the “Biological Activity” column ofTable 4) as described elsewhere herein or otherwise known in the art.Generally, variants are overall very similar, and, in many regions,identical to the amino acid sequence of the Therapeutic proteincorresponding to a Therapeutic protein portion of an albumin fusionprotein of the invention, albumin protein corresponding to an albuminprotein portion of an albumin fusion protein of the invention, and/oralbumin fusion protein of the invention. Nucleic acids encoding thesevariants are also encompassed by the invention.

The present invention is also directed to proteins which comprise, oralternatively consist of, an amino acid sequence which is at least 60%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, forexample, the amino acid sequence of a Therapeutic protein correspondingto a Therapeutic protein portion of an albumin fusion protein of theinvention (e.g., an amino acid sequence disclosed in a reference inTable 4, or fragments or variants thereof), albumin proteins (e.g.,Table 1) or fragments or variants thereof) corresponding to an albuminprotein portion of an albumin fusion protein of the invention, and/oralbumin fusion proteins of the invention. Fragments of thesepolypeptides are also provided (e.g., those fragments described herein).Further polypeptides encompassed by the invention are polypeptidesencoded by polynucleotides which hybridize to the complement of anucleic acid molecule encoding an amino acid sequence of the inventionunder stringent hybridization conditions (e.g., hybridization to filterbound DNA in 6× Sodium chloride/Sodium citrate (SSC) at about 45 degreesCelsius, followed by one or more washes in 0.2×SSC, 0.1% SDS at about50-65 degrees Celsius), under highly stringent conditions (e.g.,hybridization to filter bound DNA in 6× sodium chloride/Sodium citrate(SSC) at about 45 degrees Celsius, followed by one or more washes in0.1×SSC, 0.2% SDS at about 68 degrees Celsius), or under other stringenthybridization conditions which are known to those of skill in the art(see, for example, Ausubel, F. M. et al., eds., 1989 Current protocol inMolecular Biology, Green publishing associates, Inc., and John Wiley &Sons Inc., New York, at pages 6.3.1-6.3.6 and 2.10.3). Polynucleotidesencoding these polypeptides are also encompassed by the invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino- or carboxy-terminal positions of the reference amino acidsequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least60%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the amino acid sequence of an albumin fusion protein of theinvention or a fragment thereof (such as the Therapeutic protein portionof the albumin fusion protein or the albumin portion of the albuminfusion protein), can be determined conventionally using known computerprograms. Such programs and methods of using them are described, e.g.,in U.S. Provisional Application Ser. No. 60/355,547 and WO 01/79480 (pp.41-43), which are incorporated by reference herein, and are well knownin the art.

The polynucleotide variants of the invention may contain alterations inthe coding regions, non-coding regions, or both. Polynucleotide variantsinclude those containing alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. Such nucleotide variants may be produced bysilent substitutions due to the degeneracy of the genetic code.Polypeptide variants include those in which less than 50, less than 40,less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or1-2 amino acids are substituted, deleted, or added in any combination.Polynucleotide variants can be produced for a variety of reasons, e.g.,to optimize codon expression for a particular host (change codons in thehuman mRNA to those preferred by a microbial host, such as, yeast or E.coli).

In another embodiment, a polynucleotide encoding an albumin portion ofan albumin fusion protein of the invention is optimized for expressionin yeast or mammalian cells. In yet another embodiment, a polynucleotideencoding a Therapeutic protein portion of an albumin fusion protein ofthe invention is optimized for expression in yeast or mammalian cells.In still another embodiment, a polynucleotide encoding an albumin fusionprotein of the invention is optimized for expression in yeast ormammalian cells.

In an alternative embodiment, a codon optimized polynucleotide encodinga Therapeutic protein portion of an albumin fusion protein of theinvention does not hybridize to the wild type polynucleotide encodingthe Therapeutic protein under stringent hybridization conditions asdescribed herein. In a further embodiment, a codon optimizedpolynucleotide encoding an albumin portion of an albumin fusion proteinof the invention does not hybridize to the wild type polynucleotideencoding the albumin protein under stringent hybridization conditions asdescribed herein. In another embodiment, a codon optimizedpolynucleotide encoding an albumin fusion protein of the invention doesnot hybridize to the wild type polynucleotide encoding the Therapeuticprotein portion or the albumin protein portion under stringenthybridization conditions as described herein.

In an additional embodiment, polynucleotides encoding a Therapeuticprotein portion of an albumin fusion protein of the invention do notcomprise, or alternatively consist of, the naturally occurring sequenceof that Therapeutic protein. In a further embodiment, polynucleotidesencoding an albumin protein portion of an albumin fusion protein of theinvention do not comprise, or alternatively consist of, the naturallyoccurring sequence of albumin protein. In an alternative embodiment,polynucleotides encoding an albumin fusion protein of the invention donot comprise, or alternatively consist of, the naturally occurringsequence of a Therapeutic protein portion or the albumin proteinportion.

In an additional embodiment, the Therapeutic protein may be selectedfrom a random peptide library by biopanning, as there will be nonaturally occurring wild type polynucleotide.

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985)). These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids may be deleted from the N-terminus orC-terminus of the polypeptide of the present invention withoutsubstantial loss of biological function. See, e.g., Ron et al. (J. Biol.Chem. 268: 2984-2988 (1993) (KGF variants) and Dobeli et al., J.Biotechnology 7:199-216 (1988) (interferon gamma variants).

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein(e.g. Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993) (IL-1avariants)). Furthermore, even if deleting one or more amino acids fromthe N-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Thus, the invention further includes polypeptide variants which have afunctional activity (e.g., biological activity and/or therapeuticactivity). In further embodiments the invention provides variants ofalbumin fusion proteins that have a functional activity (e.g.,biological activity and/or therapeutic activity, such as that disclosedin the “Biological Activity” column in Table 4) that corresponds to oneor more biological and/or therapeutic activities of the Therapeuticprotein corresponding to the Therapeutic protein portion of the albuminfusion protein. Such variants include deletions, insertions, inversions,repeats, and substitutions selected according to general rules known inthe art so as have little effect on activity.

In other embodiments, the variants of the invention have conservativesubstitutions. By “conservative substitutions” is intended swaps withingroups such as replacement of the aliphatic or hydrophobic amino acidsAla, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr;replacement of the acidic residues Asp and Glu; replacement of the amideresidues Asn and Gin, replacement of the basic residues Lys, Arg, andHis; replacement of the aromatic residues Phe, Tyr, and Trp, andreplacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided, for example, in Bowie et al., “Decipheringthe Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that there are two main strategies for studying the toleranceof an amino acid sequence to change.

As the authors state, proteins are surprisingly tolerant of amino acidsubstitutions. The authors further indicate which amino acid changes arelikely to be permissive at certain amino acid positions in the protein.For example, most buried (within the tertiary structure of the protein)amino acid residues require nonpolar side chains, whereas few featuresof surface side chains are generally conserved. Moreover, toleratedconservative amino acid substitutions involve replacement of thealiphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacementof the hydroxyl residues Ser and Thr; replacement of the acidic residuesAsp and Glu; replacement of the amide residues Asn and Gln, replacementof the basic residues Lys, Arg, and His; replacement of the aromaticresidues Phe, Tyr, and Trp, and replacement of the small-sized aminoacids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acidsubstitution, variants of the present invention include (i) polypeptidescontaining substitutions of one or more of the non-conserved amino acidresidues, where the substituted amino acid residues may or may not beone encoded by the genetic code, or (ii) polypeptides containingsubstitutions of one or more of the amino acid residues having asubstituent group, or (iii) polypeptides which have been fused with orchemically conjugated to another compound, such as a compound toincrease the stability and/or solubility of the polypeptide (forexample, polyethylene glycol), (iv) polypeptide containing additionalamino acids, such as, for example, an IgG Fc fusion region peptide. Suchvariant polypeptides are deemed to be within the scope of those skilledin the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions ofcharged amino acids with other charged or neutral amino acids mayproduce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).

In specific embodiments, the polypeptides of the invention comprise, oralternatively, consist of, fragments or variants of the amino acidsequence of a Therapeutic protein described herein and/or human serumalbumin, and/or albumin fusion protein of the invention, wherein thefragments or variants have 1-5,5-10, 5-25, 5-50, 10-50 or 50-150, aminoacid residue additions, substitutions, and/or deletions when compared tothe reference amino acid sequence. In certain embodiments, the aminoacid substitutions are conservative. Nucleic acids encoding thesepolypeptides are also encompassed by the invention.

The polypeptide of the present invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result frompost-translation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Furthermore, chemical entities may be covalently attached to the albuminfusion proteins to enhance or modulate a specific functional orbiological activity such as by methods disclosed in Current Opinions inBiotechnology, 10:324 (1999).

Furthermore, targeting entities may be covalently attached to thealbumin fusion proteins of the invention to target a specific functionalor biological activity to certain cell or stage specific types, tissuetypes or anatomical structures. By directing albumin fusion proteins ofthe invention the action of the agent may be localized. Further, suchtargeting may enable the dosage of the albumin fusion proteins of theinvention required to be reduced since, by accumulating the albuminfusion proteins of the invention at the required site, a higherlocalized concentration may be achieved. Albumin fusion proteins of theinvention can be conjugated with a targeting portion by use ofcross-linking agents as well as by recombinant DNA techniques wherebythe nucleotide sequence encoding the albumin fusion proteins of theinvention, or a functional portion of it, is cloned adjacent to thenucleotide sequence of the ligand when the ligand is a protein, and theconjugate expressed as a fusion protein.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends, attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The albumin fusion proteins may also be modified with adetectable label, such as an enzymatic, fluorescent, isotopic oraffinity label to allow for detection and isolation of the protein.Examples of such modifications are given, e.g., in U.S. ProvisionalApplication Ser. No. 60/355,547 and in WO 01/79480 (pp. 105-106), whichare incorporated by reference herein, and are well known in the art.

Functional Activity

“A polypeptide having functional activity” refers to a polypeptidecapable of displaying one or more known functional activities associatedwith the full-length, pro-protein, and/or mature form of a Therapeuticprotein. Such functional activities include, but are not limited to,biological activity, enzyme inhibition, antigenicity [ability to bind toan anti-polypeptide antibody or compete with a polypeptide for binding],immunogenicity (ability to generate an antibody which binds to aspecific polypeptide of the invention), ability to form multimers withpolypeptides of the invention, and ability to bind to a receptor orligand for a polypeptide.

“A polypeptide having biological activity” refers to a polypeptideexhibiting activity similar to, but not necessarily identical to, anactivity of a Therapeutic protein of the present invention, includingmature forms, as measured in a particular biological assay, with orwithout dose dependency. In the case where dose dependency does exist,it need not be identical to that of the polypeptide, but rathersubstantially similar to the dose-dependence in a given activity ascompared to the polypeptide of the present invention.

In other embodiments, an albumin fusion protein of the invention has atleast one biological and/or therapeutic activity associated with theTherapeutic protein (or fragment or variant thereof) when it is notfused to albumin.

The albumin fusion proteins of the invention can be assayed forfunctional activity (e.g., biological activity) using or routinelymodifying assays known in the art, as well as assays described herein.Specifically, albumin fusion proteins may be assayed for functionalactivity (e.g., biological activity or therapeutic activity) using theassay referenced in the “Relevant Publications” column of Table 4.Additionally, one of skill in the art may routinely assay fragments of aTherapeutic protein corresponding to a Therapeutic protein portion of analbumin fusion protein of the invention, for activity using assaysreferenced in its corresponding row of Table 4. Further, one of skill inthe art may routinely assay fragments of an albumin proteincorresponding to an albumin protein portion of an albumin fusion proteinof the invention, for activity using assays known in the art and/or asdescribed in the Examples section below.

In addition, assays described herein (see Examples and Table 4) andotherwise known in the art may routinely be applied to measure theability of albumin fusion proteins of the present invention andfragments, variants and derivatives thereof to elicit biologicalactivity and/or Therapeutic activity (either in vitro or in vivo)related to either the Therapeutic protein portion and/or albumin portionof the albumin fusion protein of the present invention. Other methodswill be known to the skilled artisan and are within the scope of theinvention.

Expression of Fusion Proteins

The albumin fusion proteins of the invention may be produced asrecombinant molecules by secretion from yeast, a microorganism such as abacterium, or a human or animal cell line. Optionally, the polypeptideis secreted from the host cells.

For expression of the albumin fusion proteins exemplified herein, yeaststrains disrupted of the HSP150 gene as exemplified in WO 95/33833, oryeast strains disrupted of the PMT1 gene as exemplified in WO 00/44772[rHA process] (serving to reduce/eliminate O-linked glycosylation of thealbumin fusions), or yeast strains disrupted of the YAP3 gene asexemplified in WO 95/23857 were successfully used, in combination withthe yeast PRB1 promoter, the HSA/MFα-1 fusion leader sequenceexemplified in WO 90/01063, the yeast ADH1 terminator, the LEU2selection marker and the disintegration vector pSAC35 exemplified inU.S. Pat. No. 5,637,504.

Other yeast strains, promoters, leader sequences, terminators, markersand vectors which are expected to be useful in the invention aredescribed in U.S. Provisional Application Ser. No. 60/355,547 and in WO01/74980 (pp. 94-99), which are incorporated herein by reference, andare well known in the art.

The present invention also includes a cell, optionally a yeast celltransformed to express an albumin fusion protein of the invention. Inaddition to the transformed host cells themselves, the present inventionalso contemplates a culture of those cells, optionally a monoclonal(clonally homogeneous) culture, or a culture derived from a monoclonalculture, in a nutrient medium. If the polypeptide is secreted, themedium will contain the polypeptide, with the cells, or without thecells if they have been filtered or centrifuged away. Many expressionsystems are known and may be used, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae, Kluyveromyces lactis and Pichia pastoris), filamentous fungi(for example Aspergillus), plant cells, animal cells and insect cells.

The desired protein is produced in conventional ways, for example from acoding sequence inserted in the host chromosome or on a free plasmid.The yeasts are transformed with a coding sequence for the desiredprotein in any of the usual ways, for example electroporation. Methodsfor transformation of yeast by electroporation are disclosed in Becker &Guarente (1990) Methods Enzymol. 194, 182.

Successfully transformed cells, i.e., cells that contain a DNA constructof the present invention, can be identified by well known techniques.For example, cells resulting from the introduction of an expressionconstruct can be grown to produce the desired polypeptide. Cells can beharvested and lysed and their DNA content examined for the presence ofthe DNA using a method such as that described by Southern (1975) J. Mol.Biol. 98, 503 or Berent et al. (1985) Biotech. 3, 208. Alternatively,the presence of the protein in the supernatant can be detected usingantibodies.

Useful yeast plasmid vectors include pRS403-406 and pRS413-416 and aregenerally available from Stratagene Cloning Systems, La Jolla, Calif.92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are YeastIntegrating plasmids (YIps) and incorporate the yeast selectable markersHIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromereplasmids (YCps).

Vectors for making albumin fusion proteins for expression in yeastinclude pPPC0005, pScCHSA, pScNHSA, and pC4:HSA which were deposited onApr. 11, 2001 at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209 and which are described inProvisional Application Ser. No. 60/355,547 and WO 01/79480, which areincorporated by reference herein.

Another vector which is expected to be useful for expressing an albuminfusion protein in yeast is the pSAC35 vector which is described in Sleepet al., BioTechnology 8:42 (1990), which is hereby incorporated byreference in its entirety. The plasmid pSAC35 is of the disintegrationclass of vector described in U.S. Pat. No. 5,637,504.

A variety of methods have been developed to operably link DNA to vectorsvia complementary cohesive termini. For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted to thevector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. The DNAsegment, generated by endonuclease restriction digestion, is treatedwith bacteriophage T4 DNA polymerase or E. coli DNA polymerase I,enzymes that remove protruding, γ-single-stranded termini with their 3′5′-exonucleolytic activities, and fill in recessed 3′-ends with theirpolymerizing activities. The combination of these activities thereforegenerates blunt-ended DNA segments. The blunt-ended segments are thenincubated with a large molar excess of linker molecules in the presenceof an enzyme that is able to catalyze the ligation of blunt-ended DNAmolecules, such as bacteriophage T4 DNA ligase. Thus, the products ofthe reaction are DNA segments carrying polymeric linker sequences attheir ends. These DNA segments are then cleaved with the appropriaterestriction enzyme and ligated to an expression vector that has beencleaved with an enzyme that produces termini compatible with those ofthe DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of commercial sources.

A desirable way to modify the DNA in accordance with the invention, if,for example, HA variants are to be prepared, is to use the polymerasechain reaction as disclosed by Saiki et al. (1988) Science 239, 487-491.In this method the DNA to be enzymatically amplified is flanked by twospecific oligonucleotide primers which themselves become incorporatedinto the amplified DNA. The specific primers may contain restrictionendonuclease recognition sites which can be used for cloning intoexpression vectors using methods known in the art.

Exemplary genera of yeast contemplated to be useful in the practice ofthe present invention as hosts for expressing the albumin fusionproteins are Pichia (formerly classified as Hansenula), Saccharomyces,Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora,Schizosaccharomyces, Citeromyces, Pachysolen, Zygosaccharomyces,Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora,Yarrowia, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus,Sporidiobolus, Endomycopsis, and the like. Genera include those selectedfrom the group consisting of Saccharomyces, Schizosaccharomyces,Kluyveromyces, Pichia and Torulaspora. Examples of Saccharomyces spp.are S. cerevisiae, S. italicus and S. rouxii. Examples of other species,and methods of transforming them, are described in U.S. ProvisionalApplication Ser. No. 60/355,547 and WO 01/79480 (pp. 97-98), which areincorporated herein by reference.

Methods for the transformation of S. cerevisiae are taught generally inEP 251 744, EP 258 067 and WO 90/01063, all of which are incorporatedherein by reference.

Suitable promoters for S. cerevisiae include those associated with thePGKI gene, GAL1 or GAL10 genes, CYCI, PHO5, TRPI, ADHI, ADH2, the genesfor glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, triose phosphate isomerase,phosphoglucose isomerase, glucokinase, alpha-mating factor pheromone, [amating factor pheromone], the PRBI promoter, the GUT2 promoter, the GPDIpromoter, and hybrid promoters involving hybrids of parts of 5′regulatory regions with parts of 5′ regulatory regions of otherpromoters or with upstream activation sites (e.g. the promoter ofEP-A-258 067).

Convenient regulatable promoters for use in Schizosaccharomyces pombeare the thiamine-repressible promoter from the nmt gene as described byMaundrell (1990) J. Biol. Chem. 265, 10857-10864 and the glucoserepressible jbpl gene promoter as described by Hoffman & Winston (1990)Genetics 124, 807-816.

Methods of transforming Pichia for expression of foreign genes aretaught in, for example, Cregg et al. (1993), and various Phillipspatents (e.g. U.S. Pat. No. 4,857,467, incorporated herein byreference), and Pichia expression kits are commercially available fromInvitrogen BV, Leek, Netherlands, and Invitrogen Corp., San Diego,Calif. Suitable promoters include AOXI and AOX2. Gleeson et al. (1986)J. Gen. Microbiol. 132, 3459-3465 include information on Hansenulavectors and transformation, suitable promoters being MOXI and FMD1;whilst EP 361 991, Fleer et al. (1991) and other publications fromRhone-Poulenc Rorer teach how to express foreign proteins inKluyveromyces spp.

The transcription termination signal may be the 3′ flanking sequence ofa eukaryotic gene which contains proper signals for transcriptiontermination and polyadenylation. Suitable 3′ flanking sequences may, forexample, be those of the gene naturally linked to the expression controlsequence used, i.e. may correspond to the promoter. Alternatively, theymay be different in which case the termination signal of the S.cerevisiae ADHI gene is optionally used.

The desired albumin fusion protein may be initially expressed with asecretion leader sequence, which may be any leader effective in theyeast chosen. Leaders useful in S. cerevisiae include that from themating factor a polypeptide (MFα-1) and the hybrid leaders of EP-A-387319. Such leaders (or signals) are cleaved by the yeast before themature albumin is released into the surrounding medium. Further suchleaders include those of S. cerevisiae invertase (SUC2) disclosed in JP62-096086 (granted as 911036516), acid phosphatase (PH05), thepre-sequence of MFα-1,0 glucanase (BGL2) and killer toxin; S.diastaticus glucoamylase 11; S. carlsbergensis α-galactosidase (MEL1);K. lactis killer toxin; and Candida glucoamylase.

Additional Methods of Recombinant and Synthetic Production of AlbuminFusion Proteins

The present invention includes polynucleotides encoding albumin fusionproteins of this invention, as well as vectors, host cells and organismscontaining these polynucleotides. The present invention also includesmethods of producing albumin fusion proteins of the invention bysynthetic and recombinant techniques. The polynucleotides, vectors, hostcells, and organisms may be isolated and purified by methods known inthe art.

A vector useful in the invention may be, for example, a phage, plasmid,cosmid, mini-chromosome, viral or retroviral vector.

The vectors which can be utilized to clone and/or expresspolynucleofides of the invention are vectors which are capable ofreplicating and/or expressing the polynucleotides in the host cell inwhich the polynucleotides are desired to be replicated and/or expressed.In general, the polynucleotides and/or vectors can be utilized in anycell, either eukaryotic or prokaryotic, including mammalian cells (e.g.,human (e.g., HeLa), monkey (e.g., Cos), rabbit (e.g., rabbitreticulocytes), rat, hamster (e.g., CHO, NSO and baby hamster kidneycells) or mouse cells (e.g., L cells), plant cells, yeast cells, insectcells or bacterial cells (e.g., E. coli). See, e.g., F. Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley-Interscience (1992) and Sambrook et al. (1989) for examples ofappropriate vectors for various types of host cells. Note, however, thatwhen a retroviral vector that is replication defective is used, viralpropagation generally will occur only in complementing host cells.

The host cells containing these polynucleotides can be used to expresslarge amounts of the protein useful in, for example, pharmaceuticals,diagnostic reagents, vaccines and therapeutics. The protein may beisolated and purified by methods known in the art or described herein.

The polynucleotides encoding albumin fusion proteins of the inventionmay be joined to a vector containing a selectable marker for propagationin a host. Generally, a plasmid vector may be introduced in aprecipitate, such as a calcium phosphate precipitate, or in a complexwith a charged lipid. If the vector is a virus, it may be packaged invitro using an appropriate packaging cell line and then transduced intohost cells.

The polynucleotide insert should be operatively linked to an appropriatepromoter compatible with the host cell in which the polynucleotide is tobe expressed. The promoter may be a strong promoter and/or an induciblepromoter. Examples of promoters include the phage lambda PL promoter,the E. coli lac, trp, phoA and tac promoters, the SV40 early and latepromoters and promoters of retroviral LTRs, to name a few. Othersuitable promoters will be known to the skilled artisan. The expressionconstructs will further contain sites for transcription initiation,termination, and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the transcripts expressed by theconstructs may include a translation initiating codon at the beginningand a termination codon (TAA, TGA or TAG) appropriately positioned atthe end of the polypeptide to be translated.

As indicated, the expression vectors may include at least one selectablemarker. Such markers include dihydrofolate reductase, G418, glutaminesynthase, or neomycin resistance for eukaryotic cell culture, andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

In one embodiment, polynucleotides encoding an albumin fusion protein ofthe invention may be fused to signal sequences which will direct thelocalization of a protein of the invention to particular compartments ofa prokaryotic or eukaryotic cell and/or direct the secretion of aprotein of the invention from a prokaryotic or eukaryotic cell. Forexample, in E. coli, one may wish to direct the expression of theprotein to the periplasmic space. Examples of signal sequences orproteins (or fragments thereof) to which the albumin fusion proteins ofthe invention may be fused in order to direct the expression of thepolypeptide to the periplasmic space of bacteria include, but are notlimited to, the pelB signal sequence, the maltose binding protein (MBP)signal sequence, MBP, the ompA signal sequence, the signal sequence ofthe periplasmic E. coli heat-labile enterotoxin B-subunit, and thesignal sequence of alkaline phosphatase. Several vectors arecommercially available for the construction of fusion proteins whichwill direct the localization of a protein, such as the pMAL series ofvectors (particularly the pMAL-p series) available from New EnglandBiolabs. In a specific embodiment, polynucleotides albumin fusionproteins of the invention may be fused to the pelB pectate lyase signalsequence to increase the efficiency of expression and purification ofsuch polypeptides in Gram-negative bacteria. See, U.S. Pat. Nos.5,576,195 and 5,846,818, the contents of which are herein incorporatedby reference in their entireties.

Examples of signal peptides that may be fused to an albumin fusionprotein of the invention in order to direct its secretion in mammaliancells include, but are not limited to, the MPIF-1 signal sequence (e.g.,amino acids 1-21 of GenBank Accession number AAB51134), thestanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:______, anda consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:______. Asuitable signal sequence that may be used in conjunction withbaculoviral expression systems is the gp67 signal sequence (e.g., aminoacids 1-19 of GenBank Accession Number AAA72759).

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors is the availability of cell lines (e.g., themurine myeloma cell line, NSO) which are glutamine synthase negative.Glutamine synthase expression systems can also function in glutaminesynthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) byproviding additional inhibitor to prevent the functioning of theendogenous gene. A glutamine synthase expression system and componentsthereof are detailed in PCT publications: WO87/04462; WO86/05807;WO89/01036; WO89/10404; and WO91/06657, which are hereby incorporated intheir entireties by reference herein. Additionally, glutamine synthaseexpression vectors can be obtained from Lonza Biologics, Inc.(Portsmouth, N.H.). Expression and production of monoclonal antibodiesusing a GS expression system in murine myeloma cells is described inBebbington et al., Bio/technology 10:169(1992) and in Biblia andRobinson Biotechnol. Prog. 11:1 (1995) which are herein incorporated byreference.

The present invention also relates to host cells containing vectorconstructs, such as those described herein, and additionally encompasseshost cells containing nucleotide sequences of the invention that areoperably associated with one or more heterologous control regions (e.g.,promoter and/or enhancer) using techniques known of in the art. The hostcell can be a higher eukaryotic cell, such as a mammalian cell (e.g., ahuman derived cell), or a lower eukaryotic cell, such as a yeast cell,or the host cell can be a prokaryotic cell, such as a bacterial cell. Ahost strain may be chosen which modulates the expression of the insertedgene sequences, or modifies and processes the gene product in thespecific fashion desired. Expression from certain promoters can beelevated in the presence of certain inducers; thus expression of thegenetically engineered polypeptide may be controlled. Furthermore,different host cells have characteristics and specific mechanisms forthe translational and post-translational processing and modification(e.g., phosphorylation, cleavage) of proteins. Appropriate cell linescan be chosen to ensure the desired modifications and processing of theforeign protein expressed.

Introduction of the nucleic acids and nucleic acid constructs of theinvention into the host cell can be effected by calcium phosphatetransfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,or other methods. Such methods are described in many standard laboratorymanuals, such as Davis et al., Basic Methods In Molecular Biology(1986). It is specifically contemplated that the polypeptides of thepresent invention may in fact be expressed by a host cell lacking arecombinant vector.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., the coding sequence corresponding to aTherapeutic protein may be replaced with an albumin fusion proteincorresponding to the Therapeutic protein), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences such as forexample, an albumin fusion protein of the invention corresponding to theTherapeutic protein may be included). The genetic material operablyassociated with the endogenous polynucleotide may activate, alter,and/or amplify endogenous polynucleotides.

In addition, techniques known in the art may be used to operablyassociate heterologous polynucleotides (e.g., polynucleotides encodingan albumin protein, or a fragment or variant thereof) and/orheterologous control regions (e.g., promoter and/or enhancer) withendogenous polynucleotide sequences encoding a Therapeutic protein viahomologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issuedJun. 24, 1997; International Publication Number WO 96/29411;International Publication Number WO 94/12650; Koller et al., Proc. Natl.Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature342:435-438 (1989), the disclosures of each of which are incorporated byreference in their entireties).

Advantageously, albumin fusion proteins of the invention can berecovered and purified from recombinant cell cultures by well-knownmethods including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography, hydrophobic chargeinteraction chromatography and lectin chromatography. In someembodiments, high performance liquid chromatography (‘HPLC’) may beemployed for purification. In some cases, therapeutic proteins have lowsolubility or are soluble only in low or high pH or only in high or lowsalt. Fusion of therapeutic proteins to HSA is likely to improve thesolubility characteristics of the therapeutic protein.

In some embodiments albumin fusion proteins of the invention arepurified using one or more Chromatography methods listed above. In otherembodiments, albumin fusion proteins of the invention are purified usingone or more of the following Chromatography columns, Q sepharose FFcolumn, SP Sepharose FF column, Q Sepharose High Performance Column,Blue Sepharose FF column, Blue Column, Phenyl Sepharose FF column, DEAESepharose FF, or Methyl Column.

Additionally, albumin fusion proteins of the invention may be purifiedusing the process described in International Publication No. WO 00/44772which is herein incorporated by reference in its entirety. One of skillin the art could easily modify the process described therein for use inthe purification of albumin fusion proteins of the invention.

Albumin fusion proteins of the present invention may be recovered fromproducts produced by recombinant techniques from a prokaryotic oreukaryotic host, including, for example, bacterial, yeast, higher plant,insect, and mammalian cells. Depending upon the host employed in arecombinant production procedure, the polypeptides of the presentinvention may be glycosylated or may be non-glycosylated. In addition,albumin fusion proteins of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

Albumin fusion proteins of the invention and antibodies that bind aTherapeutic protein or fragments or variants thereof can be fused tomarker sequences, such as a peptide to facilitate purification. In oneembodiment, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Other peptide tags usefulfor purification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., Cell 37:767 (1984)) and the “FLAG” tag.

Further, an albumin fusion protein of the invention may be conjugated toa therapeutic moiety such as a cytotoxin, e.g., a cytostatic orcytocidal agent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. Examples of such agents aregiven in U.S. Provisional Application Ser. No. 60/355,547 and in WO01/79480 (p. 107), which are incorporated herein by reference.

Albumin fusion proteins may also be attached to solid supports, whichare particularly useful for immunoassays or purification of polypeptidesthat are bound by, that bind to, or associate with albumin fusionproteins of the invention. Such solid supports include, but are notlimited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene.

Also provided by the invention are chemically modified derivatives ofthe albumin fusion proteins of the invention which may provideadditional advantages such as increased solubility, stability andcirculating time of the polypeptide, or decreased immunogenicity (seeU.S. Pat. No. 4,179,337). Examples involving the use of polyethyleneglycol are given in WO 01/79480 (pp. 109-111), which are incorporated byreference herein.

The presence and quantity of albumin fusion proteins of the inventionmay be determined using ELISA, a well known immunoassay known in theart.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways.The following description should be considered exemplary and utilizesknown techniques.

The albumin fusion proteins of the present invention are useful fortreatment, prevention and/or prognosis of various disorders in mammals,preferably humans. Such disorders include, but are not limited to, thosedescribed herein under the heading “Biological Activity” in Table 4. Forexample, the albumin fusion proteins of the present invention may beused as inhibitors of serine proteases, plasmin, human neutrophilelastase and/or kallikrein.

Albumin fusion proteins can also be used to assay levels of polypeptidesin a biological sample. For example, radiolabeled albumin fusionproteins of the invention could be used for imaging of polypeptides in abody. Examples of assays are given, e.g., in U.S. ProvisionalApplication Ser. No. 60/355,547 and WO 0179480 (pp. 112-122), which areincorporated herein by reference, and are well known in the art. Labelsor markers for in vivo imaging of protein include, but are not limitedto, those detectable by X-radiography, nuclear magnetic resonance (NMR),electron spin relaxation (ESR), positron emission tomography (PET), orcomputer tomography (CT). For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the albumin fusion protein bylabeling of nutrients given to a cell line expressing the albumin fusionprotein of the invention.

An albumin fusion protein which has been labeled with an appropriatedetectable imaging moiety, such as a radioisotope (for example, ¹³¹I,¹¹²In, ^(99m)Tc, (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I, carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), andtechnetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F,¹⁵³Sm, ¹⁷⁷LU, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru), a radio-opaque substance, or a materialdetectable by nuclear magnetic resonance, is introduced (for example,parenterally, subcutaneously or intraperitoneally) into the mammal to beexamined for immune system disorder. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of ^(99m)Tc. The labeled albumin fusion protein will thenpreferentially accumulate at locations in the body (e.g., organs, cells,extracellular spaces or matrices) where one or more receptors, ligandsor substrates (corresponding to that of the Therapeutic protein used tomake the albumin fusion protein of the invention) are located.Alternatively, in the case where the albumin fusion protein comprises atleast a fragment or variant of a Therapeutic antibody, the labeledalbumin fusion protein will then preferentially accumulate at thelocations in the body (e.g., organs, cells, extracellular spaces ormatrices) where the polypeptides/epitopes corresponding to those boundby the Therapeutic antibody (used to make the albumin fusion protein ofthe invention) are located. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982)). The protocols described therein could easily bemodified by one of skill in the art for use with the albumin fusionproteins of the invention.

Albumin fusion proteins of the invention can also be used to raiseantibodies, which in turn may be used to measure protein expression ofthe Therapeutic protein, albumin protein, and/or the albumin fusionprotein of the invention from a recombinant cell, as a way of assessingtransformation of the host cell, or in a biological sample. Moreover,the albumin fusion proteins of the present invention can be used to testthe biological activities described herein.

Transgenic Organisms

Transgenic organisms that express the albumin fusion proteins of theinvention are also included in the invention. Transgenic organisms aregenetically modified organisms into which recombinant, exogenous orcloned genetic material has been transferred. Such genetic material isoften referred to as a transgene. The nucleic acid sequence of thetransgene may include one or more transcriptional regulatory sequencesand other nucleic acid sequences such as introns, that may be necessaryfor optimal expression and secretion of the encoded protein. Thetransgene may be designed to direct the expression of the encodedprotein in a manner that facilitates its recovery from the organism orfrom a product produced by the organism, e.g. from the milk, blood,urine, eggs, hair or seeds of the organism. The transgene may consist ofnucleic acid sequences derived from the genome of the same species or ofa different species than the species of the target animal. The transgenemay be integrated either at a locus of a genome where that particularnucleic acid sequence is not otherwise normally found or at the normallocus for the transgene.

The term “germ cell line transgenic organism” refers to a transgenicorganism in which the genetic alteration or genetic information wasintroduced into a germ line cell, thereby conferring the ability of thetransgenic organism to transfer the genetic information to offspring. Ifsuch offspring in fact possess some or all of that alteration or geneticinformation, then they too are transgenic organisms. The alteration orgenetic information may be foreign to the species of organism to whichthe recipient belongs, foreign only to the particular individualrecipient, or may be genetic information already possessed by therecipient. In the last case, the altered or introduced gene may beexpressed differently than the native gene.

A transgenic organism may be a transgenic human, animal or plant.Transgenics can be produced by a variety of different methods includingtransfection, electroporation, microinjection, gene targeting inembryonic stem cells and recombinant viral and retroviral infection(see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,602,307; Mullins etal. (1993) Hypertension 22(4):630-633; Brenin et al. (1997) Surg. Oncol.6(2)₉₉-110; Tuan (ed.), Recombinant Gene Expression Protocols, Methodsin Molecular Biology No. 62, Humana Press (1997)). The method ofintroduction of nucleic acid fragments into recombination competentmammalian cells can be by any method which favors co-transformation ofmultiple nucleic acid molecules. Detailed procedures for producingtransgenic animals are readily available to one skilled in the art,including the disclosures in U.S. Pat. No. 5,489,743 and U.S. Pat. No.5,602,307. Additional information is given in U.S. ProvisionalApplication Ser. No. 60/355,547 and WO 01/79480 (pp. 151-162), which areincorporated by reference herein.

Gene Therapy

Constructs encoding albumin fusion proteins of the invention can be usedas a part of a gene therapy protocol to deliver therapeuticallyeffective doses of the albumin fusion protein. One approach for in vivointroduction of nucleic acid into a cell is by use of a viral vectorcontaining nucleic acid, encoding an albumin fusion protein of theinvention. Infection of cells with a viral vector has the advantage thata large proportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells whichhave taken up viral vector nucleic acid. The extended plasma half-lifeof the described albumin fusion proteins may even compensate for apotentially low expression level.

Retrovirus vectors and adeno-associated virus vectors can be used as arecombinant gene delivery system for the transfer of exogenous nucleicacid molecules encoding albumin fusion proteins in vivo. These vectorsprovide efficient delivery of nucleic acids into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. Examples of such vectors, methods of using them, and theiradvantages, as well as non-viral delivery methods are described indetail in U.S. Provisional Application Ser. No. 60/355,547 and WO01/79480 (pp. 151-153), which are incorporated by reference herein.

Gene delivery systems for a gene encoding an albumin fusion protein ofthe invention can be introduced into a patient by any of a number ofmethods. For instance, a pharmaceutical preparation of the gene deliverysystem can be introduced systemically, e.g. by intravenous injection,and specific transduction of the protein in the target cells occurspredominantly from specificity of transfection provided by the genedelivery vehicle, cell-type or tissue-type expression due to thetranscriptional regulatory sequences controlling expression of thereceptor gene, or a combination thereof. In other embodiments, initialdelivery of the recombinant gene is more limited with introduction intothe animal being quite localized. For example, the gene delivery vehiclecan be introduced by catheter (see U.S. Pat. No. 5,328,470) or byStereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3054-3057). Thepharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Where the albumin fusion protein can be produced intact fromrecombinant cells, e.g. retroviral vectors, the pharmaceuticalpreparation can comprise one or more cells which produce the albuminfusion protein. Additional gene therapy methods are described in U.S.Provisional Application Ser. No. 60/355,547 and in WO 01/79480 (pp.153-162), which are incorporated herein by reference.

Pharmaceutical or Therapeutic Compositions

The albumin fusion proteins of the invention or formulations thereof maybe administered by any conventional method including parenteral (e.g.subcutaneous or intramuscular) injection or intravenous infusion. Thetreatment may consist of a single dose or a plurality of doses over aperiod of time. Furthermore, the dose, or plurality of doses, isadministered less frequently than for the Therapeutic Protein which isnot fused to albumin.

While it is possible for an albumin fusion protein of the invention tobe administered alone, it is desirable to present it as a pharmaceuticalformulation, together with one or more acceptable carriers. Thecarrier(s) must be “acceptable” in the sense of being compatible withthe albumin fusion protein and not deleterious to the recipientsthereof. Typically, the carriers will be water or saline which will besterile and pyrogen free. Albumin fusion proteins of the invention areparticularly well suited to formulation in aqueous carriers such assterile pyrogen free water, saline or other isotonic solutions becauseof their extended shelf-life in solution. For instance, pharmaceuticalcompositions of the invention may be formulated well in advance inaqueous form, for instance, weeks or months or longer time periodsbefore being dispensed.

Formulations containing the albumin fusion protein may be preparedtaking into account the extended shelf-life of the albumin fusionprotein in aqueous formulations. As discussed above, the shelf-life ofmany of these Therapeutic proteins are markedly increased or prolongedafter fusion to HA.

In instances where aerosol administration is appropriate, the albuminfusion proteins of the invention can be formulated as aerosols usingstandard procedures. The term “aerosol” includes any gas-borne suspendedphase of an albumin fusion protein of the instant invention which iscapable of being inhaled into the bronchioles or nasal passages.Specifically, aerosol includes a gas-borne suspension of droplets of analbumin fusion protein of the instant invention, as may be produced in ametered dose inhaler or nebulizer, or in a mist sprayer. Aerosol alsoincludes a dry powder composition of a compound of the instant inventionsuspended in air or other carrier gas, which may be delivered byinsufflation from an inhaler device, for example.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association the albuminfusion protein with the carrier that constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulationappropriate for the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampules, vials or syringes, and may bestored in a freeze-dried (lyophilised) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders. Dosageformulations may contain the Therapeutic protein portion at a lowermolar concentration or lower dosage compared to the non-fused standardformulation for the Therapeutic protein given the extended serumhalf-life exhibited by many of the albumin fusion proteins of theinvention.

As an example, when an albumin fusion protein of the invention comprisesone or more of the Therapeutic protein regions, the dosage form can becalculated on the basis of the potency of the albumin fusion proteinrelative to the potency of the Therapeutic protein, while taking intoaccount the prolonged serum half-life and shelf-life of the albuminfusion proteins compared to that of the native Therapeutic protein. Forexample, in an albumin fusion protein consisting of a full length HAfused to a full length Therapeutic protein, an equivalent dose in termsof units would represent a greater weight of agent but the dosagefrequency can be reduced.

Formulations or compositions of the invention may be packaged togetherwith, or included in a kit with, instructions or a package insertreferring to the extended shelf-life of the albumin fusion proteincomponent. For instance, such instructions or package inserts mayaddress recommended storage conditions, such as time, temperature andlight, taking into account the extended or prolonged shelf-life of thealbumin fusion proteins of the invention. Such instructions or packageinserts may also address the particular advantages of the albumin fusionproteins of the inventions, such as the ease of storage for formulationsthat may require use in the field, outside of controlled hospital,clinic or office conditions. As described above, formulations of theinvention may be in aqueous form and may be stored under less than idealcircumstances without significant loss of therapeutic activity.

The invention also provides methods of treatment and/or prevention ofdiseases or disorders (such as, for example, any one or more of thediseases or disorders disclosed herein) by administration to a subjectof an effective amount of an albumin fusion protein of the invention ora polynucleotide encoding an albumin fusion protein of the invention(“albumin fusion polynucleotide”) in a pharmaceutically acceptablecarrier.

Effective dosages of the albumin fusion protein and/or polynucleotide ofthe invention to be administered may be determined through procedureswell known to those in the art which address such parameters asbiological half-life, bioavailability, and toxicity, including usingdata from routine in vitro and in vivo studies such as those describedin the references in Table 4, using methods well known to those skilledin the art.

The albumin fusion protein and/or polynucleotide will be formulated anddosed in a fashion consistent with good medical practice, taking intoaccount the clinical condition of the individual patient (especially theside effects of treatment with the albumin fusion protein and/orpolynucleotide alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

For example, determining an effective amount of substance to bedelivered can depend upon a number of factors including, for example,the chemical structure and biological activity of the substance, the ageand weight of the animal, the precise condition requiring treatment andits severity, and the route of administration. The frequency oftreatments depends upon a number of factors, such as the amount ofpolynucleotide constructs administered per dose, as well as the healthand history of the subject. The precise amount, number of doses, andtiming of doses will be determined by the attending physician orveterinarian.

Albumin fusion proteins and polynucleotides of the present invention canbe administered to any animal, preferably to mammals and birds.Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep,cattle, horses and pigs, with humans being particularly preferred.

As a general proposition, the albumin fusion protein of the inventionwill be dosed lower or administered less frequently than the unfusedTherapeutic peptide. A therapeutically effective dose may refer to thatamount of the compound sufficient to result in amelioration of symptoms,disease stabilization, a prolongation of survival in a patient, orimprovement in the quality of life.

Albumin fusion proteins and/or polynucleotides can be are administeredorally, rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, gels, drops ortransdermal patch), bucally, or as an oral or nasal spray.“Pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Albumin fusion proteins and/or polynucleotides of the invention are alsosuitably administered by sustained-release systems, such as thosedescribed in U.S. Provisional Application Ser. No. 60/355,547 and WO01/79480 (pp. 129-130), which are incorporated by reference herein.

For parenteral administration, in one embodiment, the albumin fusionprotein and/or polynucleotide is formulated generally by mixing it atthe desired degree of purity, in a unit dosage injectable form(solution, suspension, or emulsion), with a pharmaceutically acceptablecarrier, i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. For example, the formulation optionally does not includeoxidizing agents and other compounds that are known to be deleterious tothe Therapeutic.

The albumin fusion proteins and/or polynucleotides of the invention maybe administered alone or in combination with other therapeutic agents.Albumin fusion protein and/or polynucleotide agents that may beadministered in combination with the albumin fusion proteins and/orpolynucleotides of the invention, include but not limited to,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, and/ortherapeutic treatments as described in U.S. Provisional Application Ser.No. 60/355,547 and WO 01/79480 (pp. 132-151) which are incorporated byreference herein. Combinations may be administered either concomitantly,e.g., as an admixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions comprising albumin fusion proteins of theinvention. Optionally associated with such container(s) can be a noticein the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

With this general description of the invention, it is believed that oneof ordinary skill in the art can, using the preceding description andthe following illustrative examples, make and utilize the alterationsdetected in the present invention and practice the claimed methods. Thefollowing working examples therefore, specifically point out differentembodiments of the present invention, and are not to be construed aslimiting in any way the remainder of the disclosure.

EXAMPLES Example 1 Construction of N-Terminal and C-TerminalAlbumin-(GGS)₄GG Linker Cloning Vectors

The recombinant albumin expression vectors pDB2243 and pDB2244 have beendescribed previously in patent application WO 00/44772. The recombinantalbumin expression vectors pAYE645 and pAYE646 have been describedpreviously in UK patent application 0217033.0. Plasmid pDB2243 wasmodified to introduce a DNA sequence encoding the 14 amino acidpolypeptide linker N-GGSGGSGGSGGSGG-C ((GGS)₄GG, “N” and “C” denote theorientation of the polypeptide sequence) (SEQ ID NO: at the C-terminalend of the albumin polypeptide in such a way to subsequently enableanother polypeptide chain to be inserted C-terminal to the (GGS)₄GGlinker to produce a C-terminal albumin fusion in the generalconfiguration, albumin-(GGS)₄GG-polypeptide. Similarly, plasmid pAYE645was modified to introduce a DNA sequence encoding the (GGS)₄GGpolypeptide linker at the N-terminal end of the albumin polypeptide insuch a way to subsequently enable another polypeptide chain to beinserted N-terminal to the (GGS)₄GG linker to produce an N-terminalalbumin fusion in the general configuration ofpolypeptide-(GGS)₄GG-albumin.

Plasmid pDB2243, described by Sleep, D., et al. (1991) Bio/Technology 9,183-187 and in patent application WO 00/44772 which contained the yeastPRB1 promoter and the yeast ADH1 terminator providing appropriatetranscription promoter and transcription terminator sequences. PlasmidpDB2243 was digested to completion with BamHI, the recessed ends wereblunt ended with T4 DNA polymerase and dNTPs, and finally religated togenerate plasmid pDB2566.

A double stranded synthetic oligonucleotide linker Bsu36I/HindIII linkerwas synthesized by annealing the synthetic oligonucleotides JH033A andJH033B. JH033A (SEQ ID NO:_(——))5-TTAGOCTTAGGTGGTTCTGGTGGTICCGGTGGTTCTGGTGG ATCCGGTGGTTAATA-3′ JH033B(SEQ ID NO:_(——)) 5′-AGCTTATTAACCACCGGATCCACCAGAACCACCGGAACGACCAGAACCACCTAAGCC-3′

The annealed Bsu36I/HindIII linker was ligated into HindIII/Bsu36I cutpDB2566 to generate plasmid pDB2575X which comprised an albumin codingregion with a (GGS)₄GG peptide linker at its C-terminal end.

Plasmid pAYE645 that contained the yeast PRB1 promoter and the yeastADH1 terminator providing appropriate transcription promoter andtranscription terminator sequences is described in UK patent application0217033.0. Plasmid pAYE645 was digested to completion with therestriction enzyme AflII and partially digested with the restrictionenzyme HindIII and the DNA fragment comprising the 3′ end of the yeastPRB1 promoter and the rHA coding sequence was isolated. Plasmid pDB2241described in patent application WO 00/44772, was digested withAflII/HindIII and the DNA fragment comprising the 5′ end of the yeastPRB1 promoter and the yeast ADH1 terminator was isolated. TheAflII/HindIII DNA fragment from pAYE645 was then cloned into theAflII/HindIII pDB2241 vector DNA fragment to create the plasmid pDB2302.Plasmid pDB2302 was digested to completion with PacI/XhoI and the 6.19kb fragment isolated, the recessed ends were blunt ended with T4 DNApolymerase and dNTPs, and religated to generate plasmid pDB2465. PlasmidpDB2465 was linearized with ClaI, the recessed ends were blunt endedwith T4 DNA polymerase and dNTPs, and religated to generate plasmidpDB2533. Plasmid pDB2533 was linearized with BlnI, the recessed endswere blunt ended with T4 DNA polymerase and dNTPs, and religated togenerate plasmid pDB2534. Plasmid pDB2534 was digested to completionwith BmgBI/BglII, the 6.96 kb DNA fragment isolated and ligated to oneof two double stranded oligonucleotide linkers, VC053NC054 andVC057/VC058 to create plasmid pDB2540, or VC055NC056 and VC057/VC058 tocreate plasmid pDB2541. VC053 (SEQ ID NO:_(——))5′-GATCTTTGGATAAGAGAGACGCTCACAAGTCCGAAGTCGCTCA CCGGT-3′ VC054 (SEQ IDNO:_(——))9 5′-pCGTTGAACCGGTGAGCGACTTCGGACTTGTGAGCGTCTCTCTTAT CCAAA-3′VC055 (SEQ ID NO:_(——)) 5′-GATCTTTGGATAAGAGAGACGCTCACAAGTCCGAAGTCGCTCATCGAT-3′ VC056 (SEQ ID NO:_(——))5′-pCCTTGAATCGATGAGCGACTTCGGACTTGTGAGCGTCTCTCTTAT CCAAA-3′ VC057 (SEQ IDNO:_(——)) 5′-pTCAAGGACCTAGGTGAGGAAAACTTCAAGGCTTTGGTCTTGATCGCTTTCGCTCAATACTTGCAACAATGTCCATTCGAAGATCAC-3′ VC058 (SEQ ID NO:_(——))5′-GTGAGCTTCGAATGGACATTGTTGCAAGTATTGAGCGAAAGCGATCAAGACCAAAGGCTTGAAGTTTTCCTCACCTAGGT-3′

A double stranded synthetic oligonucleotide linker BglII/AgeI linker wassynthesized by annealing the synthetic oligonucleotides JH035A andJH035B. JH035A (SEQ ID NO:_(——))5′-GATCTTTGGATAAGAGAGGTGGATCCGGTGGTTCCGGTGGTTCTGGTGGTTCCGGTGGTGACGCTCACAAGTCCGAAGTCGCTCA-3′ JH035B (SEQ ID NO:_(——))5′-GCGGTGAGCGACTTCGGACTTGTGAGCGTCACCACCGGAACGACCAGAACCACCGGAACCACCGGATCCACCTCTC1TATCCAAA-3′

The annealed BglII/AgeI linker was ligated into BglII/AgeI cut pDB2540to generate plasmid pDB2573X, which comprised an albumin coding regionwith a (GGS)₄GG peptide linker at its N-terminal end.

Example 2 Equilibrium Inhibition Constant for Unfused DPI-14

The amino acid sequence of DPI-14 isEAVREVCSEQAETGPCIAFFPRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSA (SEQ IDNO:______). A DNA sequence was derived from this polypeptide sequence bythe process of back-translation. The DPI-14 was expressed in Pichia andextracted from the fermentation broth supernatant using ion-exchangechromatography, hydrophobic interaction chromatography, andultrafiltration. The equilibrium inhibition constant (K_(i)) for DPI-14inhibition of human neutrophil elastase (HNE) was determined to be 15±2pM, for [HNE] 57±7 pM. The K_(i) measurement was performed using themethods set forth in Example 15.

Example 3 A Construction of N-Terminal and C-Terminal Albumin-DPI-14Fusions

The DNA sequences were provided at the 5′ or 3′ end to encode bridgingsequences between the DPI-14 coding region, the albumin coding region orthe leader sequence as appropriate for N-terminalDPI-14-(GGS)₄GG-albumin or C-terminal albumin-(GGS)₄GG-DPI-14 fusions.An N-terminal BglII-BamHI DPI-14 cDNA (Table 5) and a C-terminalBamHI-HindIII DPI-14 cDNA (Table 6) were constructed from overlappingoligonucleotides.

Example 4 Construction of N-Terminal DPI-14-(GGS)₄GG-Albumin ExpressionPlasmids

Plasmid pDB2573X was digested to completion with BglII and BamHI, the6.21 kb DNA fragment was isolated and treated with calf intestinalphosphatase and then ligated with the 0.2 kb BglII/BamHI N terminalDPI-14 cDNA to create pDB2666. The DNA and amino acid sequence of theN-terminal DPI-14(GGS)₄GG-albumin fusion are shown in Table 7 and Table8, respectively. Appropriate yeast vector sequences were provide by a“disintegration” plasmid pSAC35 generally disclosed in EP-A-286 424 anddescribed by Sleep, D., et al. (1991) Bio/Technology 9, 183-187. TheNotI N-terminal DPI-14-(GGS)₄GG-rHA expression cassette was isolatedfrom pDB2666, purified and ligated into NotI digested pSAC35 which hadbeen treated with calf intestinal phosphatase, creating two plasmids;the first (pDB2679) contained the NotI expression cassette in the sameexpression orientation as LEU2, while the second (pDB2680) contained theNotI expression cassette in the opposite orientation to LEU2. BothpDB2679 and pDB2680 are good producers of the desired fusion protein.

Example 5 Construction of C-Terminal Albumin-(GGS)₄GG-DPI-14 ExpressionPlasmid

Plasmid pDB2575X was partially digested with HindIII and then digestedto completion with BamHI. The desired 6.55 kb DNA fragment was isolatedand ligated with the 0.2 kb BamHI/HindIII C terminal DPI-14 cDNA tocreate pDB2648. The DNA and amino acid sequence of the C-terminalalbumin-(GGS)₄GG-DPI-14 fusion are shown in Table 9 and Table 10,respectively. Appropriate yeast vector sequences were provide by a“disintegration” plasmid pSAC35 generally disclosed in EP-A-286 424 anddescribed by Sleep, D., et al. (1991) Bio/Technology 9, 183-187. TheNotI C-terminal albumin-(GGS)₄GG-DPI-14 expression cassette was isolatedfrom pDB2648, purified and ligated into NotI digested pSAC35 which hadbeen treated with calf intestinal phosphatase, creating pDB2651contained the NotI expression cassette in the same expressionorientation as LEU2.

Example 6 Construction of C-Terminal Albumin-(GGS)₄GG-DX-1000 ExpressionPlasmid

Plasmid pDB2575X was partially digested with HindIII and then digestedto completion with BamHI. The desired 6.55 kb DNA fragment was isolatedand ligated with the 0.2 kb BamHI/HindIII C-terminal DX-1000 cDNA asshown in Table 11 to create pDB2648X-1000. Appropriate yeast vectorsequences were provide by a “disintegration” plasmid pSAC35 generallydisclosed in EP-A-286 424 and described by Sleep, D., et al. (1991)Bio/Technology 9, 183-187. The NotI C-terminal albumin-(GGS)₄GG-DX1000expression cassette was isolated from pDB2648X-1000, purified andligated into NotI digested pSAC35 which had been treated with calfintestinal phosphatase, creating pDB2651X-1000 contained the NotIexpression cassette in the same expression orientation as LEU2.

Example 7 Construction of N-Terminal and C-Terminal Albumin-DX-890Fusions

Generation of the Basic Clone

The amino acid sequence of DX-890 isEACNLPIVRGPCIAFFPRWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECREYCGVP (SEQ IDNO:______). A DNA sequence was derived from this polypeptide sequence bythe process of back-translation. The DNA sequences were provided at the5′ or 3′ end to encode bridging sequences between the DX-890 codingregion, the albumin coding region or the leader sequence as appropriatefor N-terminal DX-890-(GGS)₄GG-albumin or C-terminalalbumin-(GGS)₄GG-DX-890 fusions. An N-terminal Bgm-BamHI DX-890 cDNA(Table 12) and a C-terminal BamHI-HindIII DX-890 cDNA (Table 13) wereconstructed from overlapping oligonucleotides.

Example 8 Construction of N-Terminal DX-890-(GGS)₄GG-Albumin ExpressionPlasmids

Plasmid pDB2573X was digested to completion with BglII and BamHI, the6.21 kb DNA fragment was isolated and treated with calf intestinalphosphatase and then ligated with the 0.2 kb BglII/BamHI N terminalDX-890 cDNA to create pDB2683. The DNA and amino acid sequence of theN-terminal DX-890-(GGS)₄GG-albumin fusion are shown in Table 14 andTable 15, respectively. Appropriate yeast vector sequences were provideby a “disintegration” plasmid pSAC35 generally disclosed in EP-A-286 424and described by Sleep, D., et al. (1991) Bio/Technology 9, 183-187. TheNotI N-terminal DX-890-(GGS)₄GG-rHA expression cassette was isolatedfrom pDB2683, purified and ligated into NotI digested pSAC35 which hadbeen treated with calf intestinal phosphatase creating pDB2684 containedthe NotI expression cassette in the opposite orientation to LEU2.

Example 9 Construction of C-Terminal Albumin-(GGS)₄GG-DX-890 ExpressionPlasmid

Plasmid pDB2575X was partially digested with HindIII and then digestedto completion with BamHI. The desired 6.55 kb DNA fragment was isolatedand ligated with the 0.2 kb BamHI/HindIII C terminal DX-890 cDNA tocreate pDB2649. The DNA and amino acid sequence of the C-terminalalbumin-(GGS)₄GG-DX-890 fusion are shown in Table 16 and Table 17,respectively. Appropriate yeast vector sequences were provide by a“disintegration” plasmid pSAC35 generally disclosed in EP-A-286 424 anddescribed by Sleep, D., et al. (1991) Bio/Technology 0.9, 183-187. TheNotI C-terminal albumin-(GGS)₄GG-DX-890 expression cassette was isolatedfrom pDB2649, purified and ligated into NotI digested pSAC35 which hadbeen treated with calf intestinal phosphatase, creating two plasmids;the first pDB2652 contained the NotI expression cassette in the sameexpression orientation as LEU2, while the second pDB2653 contained theNotI expression cassette in the opposite orientation to LEU2.

Example 10 Fermentation to Produce a Fusion Protein

The DX-890-HSA fusion-protein was expressed in fermentation culture asdescribed in WO 00/44772. The DX-890-HSA fusion protein was purifiedfrom fermentation culture supernatant using the standard HA purificationSP-FF (Pharmacia) conditions as described in WO 00/44772, except that anextra 200 mM NaCl was required in the elution buffer.

Example 11 Yeast Transformation and Culturing Conditions

Yeast strains disclosed in WO 95/23857, WO 95/33833 and WO 94/04687 weretransformed to leucine prototrophy as described in Sleep D., et al.(2001) Yeast 18, 403-421. The transformants were patched out ontoBuffered Minimal Medium (BMM, described by Kerry-Williams, S. M. et al.(1998) Yeast 14, 161-169) and incubated at 30° C. until grownsufficiently for further analysis.

Example 12 Ki Measurement of DX-890 Samples

Equilibrium inhibition constants (K_(i)) for DX-890 or DX-890-HSAinhibition of HNE were determined according to the tight-bindinginhibition model with formation of a reversible complex (1:1stoichiometry). Inhibition of hNE was determined at 30° C. in 50 mMHEPES, pH 7.5, 150 mM NaCl, and 0.1% Triton X-100. All reactions (totalvolume=200 μL) were carried out in microtiter plates (Costar #3789). hNEwas incubated with varying concentrations of added inhibitor for 24hours. Residual enzymatic activities were determined from the relativerates of substrate hydrolysis. The hydrolysis reaction was initiated byaddition of N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amino-methylcoumarin assubstrate. Enzymatic cleavage of this substrate releases themethylcoumarin moiety with concomitant increase the sample fluorescence.The rate of substrate hydrolysis was monitored at an excitation of 360nm and an emission of 460 nm. Plots of the percent remaining activityversus inhibitor concentration were fit by nonlinear regression analysisto Equation 1 to determine equilibrium dissociation constants.$\begin{matrix}{{\%\quad A} = {100 - {\left( \frac{\left( {I + E + K_{i}} \right) - \sqrt{\left( {I + E + K_{i}} \right)^{2} - {4 \cdot E \cdot I}}}{2 \cdot E} \right) \cdot 100}}} & (1)\end{matrix}$Where:

-   % A=percent activity-   I=DX-890-   E=HNE concentration-   K_(i)=equilibrium inhibition constant

The K_(i) of native DX-890 was measured at the same time as a positivecontrol. The K_(i)'s of DX-890 and DX-890-HSA fusion for humanneutrophil elastase (HNE) were similar to each other (FIG. 1). Similarresults were seen with the DX-890-HSA fusion in supernatant from a shakeflask yeast culture or from a fermentor. Both supernatants were suppliedby Aventis to Dyax. This result indicates that fusion to HSA does notaffect the potency of DX-890 as an inhibitor of HNE.

Example 13 Fusions of DX-88 to N Terminus of HSA

DX-88 is a Kunitz domain derived from the first Kunitz domain of humanLACI which inhibits human plasma kallikrein with Ki ˜40 pM. The serumhalf-time of DX-88 is not more than 1 hour. DX-88 is currently beingtested in the clinic for treatment of hereditary angioedema (HAE).Initial data suggest that DX-88 is safe and effective. HAE is acondition in which attacks recur episodically and having a long-actingform would allow prophylactic treatment instead of reactive treatment.

A DNA sequence is available for DX-88, prepared for fusion to the Nterminus of HA. The DNA sequences are provided at the 5′ or 3′ end toencode bridging sequences between the DX-88 coding region, the albumincoding region or the leader sequence as appropriate for N-terminalDX-88-(GGS)₄GG-albumin (Table 18).

Plasmid pDB2573X is digested to completion with BglII and BamHI, the6.21 kb DNA fragment is isolated and treated with calf intestinalphosphatase and then ligated with the 0.2 kb BglII/BamHI N terminalDX-88 cDNA to create pDB2666-88. The DNA and amino acid sequence of theN-terminal DX-88-(GGS)₄GG-albumin fusion are shown in Table 19 and Table20, respectively. Appropriate yeast vector sequences are provided by a“disintegration” plasmid pSAC35 generally disclosed in EP-A-286 424 anddescribed by Sleep, D., et al. (1991) Bio/Technology 9, 183-187. TheNotI N-terminal DX-88-(GGS)₄GG-rHA expression cassette is isolated frompDB2666-88, purified and ligated into NotI digested pSAC35 which hadbeen treated with calf intestinal phosphatase, creating two plasmids;the first pDB2679-88 contains the NotI expression cassette in the sameexpression orientation as LEU2, while the second pDB2680-88 contains theNotI expression cassette in the opposite orientation to LEU2.

Example 14 Construction of C-Terminal Albumin-(GGS)₄GG-DX-88 ExpressionPlasmid

As in Example 5, Plasmid pDB2575X is partially digested with HindIII andthen digested to completion with BamHI. The desired 6.55 kb DNA fragmentis isolated and ligated with the 0.2 kb BamHI/HindIII C terminal DX-88cDNA (Table 21) to create pDB2648-88. The DNA and amino acid sequence ofthe C-terminal albumin-(GGS)₄GG-DX-88 fusion are shown in Table 22 andTable 23, respectively. Appropriate yeast vector sequences are provideby a “disintegration” plasmid pSAC35 generally disclosed in EP-A-286 424and described by Sleep, D., et al. (1991) Bio/Technology 9, 183-187. TheNotI C-terminal albumin-(GGS)₄GG-DX-88 expression cassette is isolatedfrom pDB2648-88, purified and ligated into NotI digested pSAC35 which istreated with calf intestinal phosphatase, creating pDB2651-88 containedthe NotI expression cassette in the same expression orientation as LEU2.

Example 15 Pharmacokinetic Study in Mice

The DX-890-HSA fusion protein was expressed in fermentation culture asdescribed in WO 00/44772. The DX-890-HSA fusion protein was purifiedfrom fermentation culture supernatant using the standard HA purificationSP-FF (Pharmacia) conditions as described in WO 00/44772, except that anextra 200 mM NaCl was required in the elution buffer.

About 10 mg of rHA-DX-890 fusion was purified from the diafiltrationretentate by SEC-HPLC and characterized by SCS-PAGE and RP-HPLC methodsto be about 92% monomeric form. This material was used for subsequent¹²⁵I radiolabeling and in-vivo plasma clearance studies.

For studies using mice, animals were injected in the tail vein and 4animals were sacrificed at approximately 0, 7, 15, 30 and 90 minutes,4h, 8h, 16h, 24h after injection, less 4 time points for the nativeDX-890 because of its likely short half life. Time of injection and timeof sampling were recorded. At sacrifice, samples of ˜0.5 ml werecollected into anticoagulant (0.02 ml EDTA). Cells were spun down andseparated from plasma. Plasma was divided into two aliquots, one frozenand one stored at 4° C. for immediate analysis. Analysis included gammacounting of all samples. In addition, analysis was performed for twoplasma samples (N=2) at each time point, i.e., 0, and 30 minutes, for¹²⁵I-DX-890, and 0, 30 minutes, and 24 h for the ¹²⁵I-DX-890-HSA fusion.A SEC −HPLC Superose-12 column with an in-line radiation detector wasused to analyze plasma fractions.

The results show that fusing DX-890 to HSA dramatically improves itsbeta (elimination) half life by ˜5× (FIG. 2). In addition, it appearsthat the DX-890-HSA-fusion is more stable in mouse plasma than DX-890(FIGS. 3 and 4).

Example 16 Pharmacokinetic Study in Rabbits

Pharmacokinetic properties of DX-890 and DX-890-HSA were measured byiodinating the proteins and measuring clearance of the radiolabel fromcirculation in rabbits. The two DX-890 preparations were iodinated withiodine-125 using the iodogen method. After radiolabeling, the twolabeled protein preparations were purified from unbound label by sizeexclusion chromatography (SEC). Fractions from the SEC column having thehighest radioactivity were pooled. The purified, radiolabeledpreparations were characterized for specific activity by scintillationcounting and for purity by SEC using a Superose-12 column equipped withan in-line radiation detector.

New Zealand White rabbits (ca. 2.5 Kg) were used for clearancemeasurements, with one animal each used for of the two labeled proteinpreparations. The radiolabeled preparation was injected into the animalvia an ear vein. One blood sample was collected per animal per timepoint with early time points at approximately 0, 7, 15, 30, and 90minutes and later time points at 4, 8, 16, 24, 48, 72, 96, 144, 168, and192 hours. Samples (about 0.5 ml) were collected into anticoagulant(EDTA) tubes. Cells were separated from the plasma/serum fraction bycentrifugation. The plasma fraction was divided into two aliquots. Oneplasma aliquot was stored at −70° C. and the other aliquot was kept at4° C. for immediate analyses. Sample analyses included radiationcounting for clearance rate determinations and SEC chromatography for invivo stability. The results of the rabbit clearance study are summarizedin FIGS. 5 and 6 and in Table 24.

The HSA-DX-890 fusion protein shows substantial improvements in in vivocirculation properties relative to those of the unmodified DX-890.Plasma clearance rates are greatly reduced for the fusion protein sothat after a single day relative circulating levels of radiolabel aremore than 100-fold higher for the HSA-DX-890 fusion than for theunmodified protein (FIG. 5). A simple bi-exponential fit to the datashows large increases in both the alpha and beta portions of theclearance curve (Table 24). In particular, the value for T_(1/2β) isincreased more than 20-fold, from about 165 min (2.75 hrs) for theunmodified protein to about 3500 min (˜60 hrs, ˜2.5 days) for theHSA-DX-890 fusion. In addition, the fraction of the total materialinvolved in the slow clearance portion of the curve nearly doubles forthe fusion protein relative to unmodified DX-890 (Table 24). TABLE 24Clearance Times in Rabbits Dose Clearance Times (min) Compound μgm μCiT_(1/2α) % α T_(1/2β) % β DX-890 50 83 0.4 75 165 25 MSA-DX-890 151 105270 60 3500 40

Finally, in vivo stability appears to be improved for the fusion proteinrelative to unmodified DX-890 (FIG. 6). SEC analysis of plasma from therabbit injected with ¹²⁵I-DX-890 (FIG. 6, Part A) shows a relativelyrapid association of label with higher molecular weight plasmacomponents (earlier eluting peaks). Further, the relative proportion ofthe total residual circulating label associated with the high molecularweight material increases as time post-injection increases (compare 30min and 4 hour elution profiles). In contrast, SEC analyses of plasmasamples from the rabbit injected with ¹²⁵I-HSA-DX-890 (FIG. 6, Part B)shows that almost all of the circulating label is associated with theHSA-DX-890 peak seen in the injectate and that the label remains stablyassociated with this peak for at least 72 hours.

Example 17 A Vector for Making a Doubly Fused HSA

The vector pDB2300X1 is a modification of pDB2575X in which there is aBglII/BamHI cassette near the 5′ terminus of the rHA gene and aBspEI/KpnI cassette near the 3′ terminus. The NotI cassette thatcomprises this gene is shown in Table 25 showing the DNA, encoded AAsequence and useful restriction sites. In each line in Table 25,everything after an exclamation point is commentary, the DNA sequence isnumbered and spaced to allow understand the design.

Example 18 Adding a First Instance of DX890 to PDB2300X1

The DNA shown in Table 12 is introduced into pDB2300X1 that has been cutwith BglII and BamHI to make the new vector pDB2300×2. The DNA, encodedAA sequence and useful restriction sites of the NotI cassette ofpDB2300X2 are shown in Table 26.

Example 19 Adding a Second Instance of DX890 to pDB2300X2

The DNA shown in Table 27 is introduced into pDB2300X2 that has been cutwith BspEI and KpnI to make the new vector pDB2300X3. Although this DNAencodes the same AA sequence as does the DNA of Table 12, many codonshave been changed to reduce the likelihood of recombination between thetwo DX890-encoding regions. The DNA, encoded AA sequence and usefulrestriction sites of this construct are shown in Table 28. The encodedAA sequence is shown in Table 29. This protein is expressed in the samemanner as the other constructions of the present invention. The proteinof Table 103, “Dx890-HA-Dx890”, will have ˜16% the HNE-neutralizingactivity of DX890 but a much long serum life time. Thusarea-under-the-curve for inhibition of HNE will be much higher than fornaked DX890.

Example 20 DX1000::(GGS)₄GG::HSA

The DNA shown in Table 30 is introduced into pDB2573X which has been cutwith BglII and BamHI to create pDX1000. The AA sequence of the encodedprotein is shown in Table 31. Expression of this protein is essentiallythe same as for other HA fusions of the present invention.

Example 21 DX-88::(GGS)₄GG::HSA::(GGS)₄GG::DX-88

In a manner similar to the construction of a gene encodingDX-890-HSA-DX-890, the DNA of Table 18 is inserted into pDB2300X1 thathas been cut with BglII and BamHI to make the new vector pDB2300×88a.The DNA shown in Table 32 is introduced into pDB2300×88a as a BspEI/KpnIfragment to create pDB2300×88b which contains two instances of DNA thatencodes DX-88. The DNA in Table 32 is substantially different from theDNA in Table 18 so that recombination is unlikely.

Example 22 Multiple Albumin Fusions

The N-terminal fusion expression plasmid, pDB2540, as described herein,can be modified to introduce a unique Bsu36I at the C-terminal end; thenew plasmid is named pDB2301X. The DNA sequence of the NotI expressioncassette from pDB2301X is as follows: pDB254O+Bsu36I   NotI 1 GCGGCCGCccgtaatgcggt atcgtgaaag cgaaaaaaaa actaacagta gataagacag 61 atagacagatagagatggac gagaaacagg gggggagaaa aggggaaaag agaaggaaag                                           NarI 121 aaagactcatctatcgcaga taagacaatc aaccctcatG GCGCCtccaa ccaccatccg 181 cactagggaccaagcgctcg caccgttagc aacgcttgac tcacaaacca actgccggct 241 gaaagagcttgtgcaatggg agtgccaatt caaaggagcc gaatacgtct gctcgccttt 301 taagaggctttttgaacact gcattgcacc cgacaaatca gccactaact acgaggtcac 361 ggacacatataccaatagtt aaaaattaca tatactctat atagcacagt agtgtgataa 421 ataaaaaattttgccaagac ttttttaaac tgcacccgac agatcaggtc tgtgcctact 481 atgcacttatgcccggggtc ccgggaggag aaaaaacgag ggctgggaaa tgtccgtgga 541 ctttaaacgctccgggttag cagagtagca gggctttcgg ctttggaaat ttaggtgact 601 tgttgaaaaagcaaaatttg ggctcagtaa tgccactgca gtggcttatc acgccaggac 661 tgcgggagtggcgggggcaa acacacccgc gataaagagc gcgatgaata taaaaggggg 721 ccaatgttacgtcocgttat attggagttc ttcccataca aacttaagag tccaattagc                    HindIII 781 ttcatcgcca ataaaaaaac AAGCTTaacctaattctaac aagcaaagat gaagtgggtt                                                     >>..........>                                              BglII 841 ttcatcgtctccattttgtt cttgttctcc tctgcttact ctAGATCTttggataagaga >........................FusionLeader.........................>>                             AgeI 901gacgctcaca agtccgaagt cgctcACCGG Ttcaaggacc taggtgaggaaaacttcaag >>                  rHA synth. gene ..Continues to base2655......> 961 gctttggtct tgatcgcttt cgctcaatac ttgcaacaat gtccattcgaagatcacgtc 1021 aagttggtca acgaagttac cgaattcgct aagacttgtg ttgctgacgaatctgctgaa 1081 aactgtgaca agtccttgca caccttgttc ggtgataagt tgtgtactgttgctaccttg 1141 agagaaacct acggtgaaat ggctgactgt tgtgctaagc aagaaccagaaagaaacgaa 1201 tgtttcttgc aacacaagga cgacaaccca aacttgccaa gattggttagaccagaagtt 1261 gacgtcatgt gtactgcttt ccacgacaac gaagaaacct tcttgaagaagtacttgtac 1321 gaaattgcta gaagacaccc atacttctac gctccagaat tgttgttcttcgctaagaga 1381 tacaaggctg ctttcaccga atgttgtcaa gctgctgata aggctgcttgtttgttgcca 1441 aagttggatg aattgagaga cgaaggtaag gcttcttccg ctaagcaaagattgaagtgt 1501 gcttccttgc aaaagttcgg tgaaagagct ttcaaggctt gggctgtcgctagattgtct 1561 caaagattcc caaaggctga attcgctgaa gtttctaagt tggttactgacttgactaag 1621 gttcacactg aatgttgtca cggtgacttg ttggaatgtg ctgatgacagagctgacttg 1681 gctaagtaca tctgtgaaaa ccaagactct atctcttcca agttgaaggaatgttgtgaa 1741 aagccattgt tggaaaagtc tcactgtatt gctgaagttg aaaacgatgaaatgccagct 1801 gacttgccat ctttggctgc tgacttcgtt gaatctaagg acgtttgtaagaactacgct 1861 gaagctaagg acgtcttctt gggtatgttc ttgtacgaat acgctagaagacacccagac 1921 tactccgttg tcttgttgtt gagattggct aagacctacg aaactaccttggaaaagtgt 1981 tgtgctgctg ctgacccaca cgaatgttac gctaaggttt tcgatgaattcaagccattg 2041 gtcgaagaac cacaaaactt gatcaagcaa aactgtgaat tgttcgaacaattgggtgaa 2101 tacaagttcc aaaacgcttt gttggttaga tacactaaga aggtcccacaagtctccacc 2161 ccaactttgg ttgaagtctc tagaaacttg ggtaaggtcg gttctaagtgttgtaagcac 2221 ccagaagcta agagaatgcc atgtgctgaa gattacttgt ccgtcgttttgaaccaattg 2281 tgtgttttgc acgaaaagac cccagtctct gatagagtca ccaagtgttgtactgaatct 2341 ttggttaaca gaagaccatg tttctctgct ttggaagtcg acgaaacttacgttccaaag                                     EcoRV 2401 gaattcaacgctgaaacttt caccttccac gctGATATCt gtaccttgtc cgaaaaggaa 2461 agacaaattaagaagcaaac tgctttggtt gaattggtca agcacaagcc aaaggctact 2521 aaggaacaattgaaggctgt catggatgat ttcgctgctt tcgttgaaaa gtgttgtaag 2581 gctgatgataaggaaacttg tttcgctgaa gaaggtaaga agttggtcgc tgcttcccaa     Bsu36I         HindIII 2641 gctgCCTTAG GcttataatA AGCTTaattcttatgattta tgatttttat tattaaataa >             >> 2701 gttataaaaaaaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaaaa 2761 ttcttattcttgagtaactc tttcctgtag gtcaggttgC tttctcaggt atagcatgag                               SphI 2821 gtcgctctta ttgaccacacctctaccgGC ATGCcgagca aatgcctgca aatcgctccc 2881 catttcaccc aattgtagatatgctaactc cagcaatgag ttgatgaatc tcggtgtgta                                                           NotI 2941ttttatgtcc tcagaggaca acacctgttg taatcgttct tccacacgga tcGCGGCCGCDNA encoding polypeptides can be inserted in between the BglII and AgeIsites to express an N-terminal albumin fusion, or between the Bsu36I andHindIII (not unique and so will require a partial HindIII digest) sitesto express an C-terminal albumin fusion, or between both pairs of sitesto make a co-N- and C-terminal albumin fusion.

Polypeptide spacers can be optionally incorporated. The DNA sequence ofthe NotI expression cassette from the modified pDB2540 is expected to beas follows: pDB2540+2xGSlinkers   NotI 1 GCGGCCGCcc gtaatgcggtatcgtgaaag cgaaaaaaaa actaacagta gataagacag 61 atagacagat agagatggacgagaaacagg gggggagaaa aggggaaaag agaaggaaag                                           NarI 121 aaagactcatctatcgcaga taagacaatc aaccctcatG GCGCCtccaa ccaccatccg 181 cactagggaccaagcgctcg caccgttagc aacgcttgac tcacaaacca actgccggct 241 gaaagagcttgtgcaatggg agtgccaatt caaaggagcc gaatacgtct gctcgccttt 301 taagaggctttttgaacact gcattgcacc cgacaaatca gccactaact acgaggtcac 361 ggacacatataccaatagtt aaaaattaca tatactctat atagcacagt agtgtgataa 421 ataaaaaattttgccaagac ttttttaaac tgcacccgac agatcaggtc tgtgcctact 481 atgcacttatgcccggggtc ccgggaggag aaaaaacgag ggctgggaaa tgtccgtgga 541 ctttaaacgctccgggttag cagagtagca gggctttcgg ctttggaaat ttaggtgact 601 tgttgaaaaagcaaaatttg ggctcagtaa tgccactgca gtggcttatc acgccaggac 661 tgcgggagtggcgggggcaa acacacccgc gataaagagc gcgatgaata taaaaggggg 721 ccaatgttacgtcccgttat attggagttc ttcccataca aacttaagag tccaattagc                     HindIII 781 ttcatcgcca ataaaaaaac AAGCTTaacctaattctaac aagcaaagat gaagtgggtt                                                    >>..........>                                              BglII 841 ttcatcgtctccattttgtt cttgttctcc tctgcttact ctAGATCTttggataagaga >........................FusionLeader.........................    BamHI 901 ggtGGATCCg gtggttccggtggttctggt ggttccggtg gtgacgctca caagtccgaa >>................GSlinker.................>|>>.....rHA........>         AgeI 961 gtcgctcACCGGTtcaagga cctaggtgag gaaaacttca aggctttggtcttgatcgCt >..............rHA synth. gene continues to base2739.............> 1021 ttcgctcaat acttgcaaca atgtccattc gaagatcacgtcaagttggt caacgaagtt 1081 accgaattcg ctaagacttg tgttgctgac gaatctgctgaaaactgtga caagtccttg 1141 cacaccttgt tcggtgataa gttgtgtact gttgctaccttgagagaaac ctacggtgaa 1201 atggctgact gttgtgctaa gcaagaacca gaaagaaacgaatgtttctt gcaacacaag 1261 gacgacaacc caaacttgcc aagattggtt agaccagaagttgacgtcat gtgtactgct 1321 ttccacgaca acgaagaaac cttcttgaag aagtacttgtacgaaattgc tagaagacac 1381 ccatacttct acgctccaga attgttgttc ttcgctaagagatacaaggc tgctttcacc 1441 gaatgttgtc aagctgctga taaggctgct tgtttgttgccaaagttgga tgaattgaga 1501 gacgaaggta aggcttcttc cgctaagcaa agattgaagtgtgcttcctt gcaaaagttc 1561 ggtgaaagag ctttcaaggc ttgggctgtc gctagattgtctcaaagatt cccaaaggct 1621 gaattcgctg aagtttctaa gttggttact gacttgactaaggttcacac tgaatgttgt 1681 cacggtgact tgttggaatg tgctgatgac agagctgacttggctaagta catctgtgaa 1741 aaccaagact ctatctcttc caagttgaag gaatgttgtgaaaagccatt gttggaaaag 1801 tctcactgta ttgctgaagt tgaaaacgat gaaatgccagctgacttgcc atctttggct 1861 gctgacttcg ttgaatctaa ggacgtttgt aagaactacgctgaagctaa ggacgtcttc 1921 ttgggtatgt tcttgtacga atacgctaga agacacccagactactccgt tgtcttgttg 1981 ttgagattgg ctaagaccta cgaaactacc ttggaaaagtgttgtgctgc tgctgaccca 2041 cacgaatgtt acgctaaggt tttcgatgaa ttcaagccattggtcgaaga accacaaaac 2101 ttgatcaagc aaaactgtga attgttcgaa caattgggtgaatacaagtt ccaaaacgct 2161 ttgttggtta gatacactaa gaaggtccca caagtctccaccccaacttt ggttgaagtc 2221 tctagaaact tgggtaaggt cggttctaag tgttgtaagcacccagaagc taagagaatg 2281 ccatgtgctg aagattactt gtccgtcgtt ttgaaccaattgtgtgtttt gcacgaaaag 2341 accccagtct ctgatagagt caccaagtgt tgtactgaatctttggttaa cagaagacca 2401 tgtttctctg ctttggaagt cgacgaaact tacgttccaaaggaattcaa cgctgaaact                  EcoRV 2461 ttcaccttCC acgctGATATCtgtaccttg tccgaaaagg aaagacaaat taagaagcaa 2521 actgctttgg ttgaattggtcaagcacaag ccaaaggcta ctaaggaaca attgaaggct 2581 gtcatggatg atttcgctgctttcgttgaa aagtgttgta aggctgatga taaggaaact                                                   Bsu36I 2641tgtttcgctg aagaaggtaa gaagttggtc gctgcttccc aagctgCCTTAGGcttaggt >.....................rHA synth.gene......................>|>>>             BspEI           KpnI              HindIII 2701 ggttctggtggtTCCGGAgg ttctggtGGT ACCggtggtt aatAAGCTTaattcttatga >...............GS linker...............>> 2761 tttatgatttttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt 2821 gactcttaggttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg                                                          SphI 2881ttgctttctc aggtatagca tgaggtcgct cttattgacc acacctctac cgGCATGCcg 2941agcaaatgcc tgcaaatcgc tccccatttc acccaattgt agatatgcta actccagcaa 3001tgagttgatg aatctcggtg tgtattttat gtcctcagag gacaacacct gttgtaatcg                   Not I 3061 ttcttccaca cggatcGCGG CCGC

DNA encoding polypeptides can be inserted in between the BglII and BamHIsites to express an N-terminal albumin fusion, or between the uniqueBspEI and KpnI sites to express an C-terminal albumin fusion, or betweenboth pairs of sites to make a co-N- and C-terminal albumin fusion. Thisis exemplified most simply by using the Bgm-BamHI DPI-14 cDNA and theBamHI-HindIII DX-890 cDNA as described herein. By ligating these cDNAsinto the appropriate site, a DPI-14-(GGS)₄GG-rHA-(GGS)₄GG-DX-890 fusionwith the following DNA sequence would be constructed.   Not I 1GCGGCCGCcc gtaatgcggt atcgtgaaag cgaaaaaaaa actaacagta gataagacag 61atagacagat agagatggaC gagaaacagg gggggagaaa aggggaaaag agaaggaaag                                           NarI 121 aaagactcatctatcgcaga taagacaatc aaccctcatG GCGCCtccaa ccaccatccg 181 cactagggaccaagcgctcg caccgttagc aacgcttgac tcacaaacca actgccggct 241 gaaagagcttgtgcaatggg agtgccaatt caaaggagcc gaatacgtct gctcgccttt 301 taagaggctttttgaacact gcattgcacc cgacaaatca gccactaact acgaggtcac 361 ggacacatataccaatagtt aaaaattaca tatactctat atagcacagt agtgtgataa 421 ataaaaaattttgccaagac ttttttaaac tgcacccgac agatcaggtc tgtgcctact 481 atgcacttatgcccggggtc ccgggaggag aaaaaacgag ggctgggaaa tgtccgtgga 541 ctttaaacgctccgggttag cagagtagca gggctttcgg ctttggaaat ttaggtgact 601 tgttgaaaaagcaaaatttg ggctcagtaa tgccactgca gtggcttatc acgccaggac 661 tgcgggagtggcgggggcaa acacacccgc gataaagagc gcgatgaata taaaaggggg 721 ccaatgttacgtcccgttat attggagttc ttcccataca aacttaagag tccaattagc                     HindIII 781 ttcatcgcca ataaaaaaac AAGCTTaacctaattctaac aagcaaagat gaagtgggtt                                                    >>..........>                                              BglII 841 ttcatcgtctccattttgtt cttgttctcc tctgcttact ctAGATCTttggataagaga >........................FusionLeader.........................>> 901 gaagctgtta gagaagtttg ttctgaacaagctgaaactg gtccatgtat tgctttcttc >>......................DPI-14 up tobase 1080...................> 961 ccaagatggt acttcgatgt tactgaaggtaagtgcgcgc cattcttcta cggtggttgt 1021 ggtggtaaca gaaacaactt cgatactgaagaatactgta tggctgtttgtggttctgct >............................DPI-14............................>>   BamHI 1081 ggtGGATCCg gtggttccgg tggttctggt ggttccggtg gtgacgctcacaagtccgaa >>................GS linker.................>|>>...rHA synthgene.>         AgeI 1141 gtcgctcACC GGTtcaagga cctaggtgag gaaaacttcaaggctttggt cttgatcgCt >.............rHA synth. gene continues to base2877..............> 1201 ttcgctcaat acttgcaaca atgtccattc gaagatcacgtcaagttggt caacgaagtt 1261 accgaattcg ctaagacttg tgttgctgac gaatctgctgaaaactgtga caagtccttg 1321 cacaccttgt tcggtgataa gttgtgtact gttgctaccttgagagaaac ctacggtgaa 1381 atggctgact gttgtgctaa gcaagaacca gaaagaaacgaatgtttctt gcaacacaag 1441 gacgacaacc caaacttgcc aagattggtt agaccagaagttgacgtcat gtgtactgct 1501 ttccacgaca acgaagaaac cttcttgaag aagtacttgtacgaaattgc tagaagacac 1561 ccatacttct acgctccaga attgttgttc ttcgctaagagatacaaggc tgctttcacc 1621 gaatgttgtc aagctgctga taaggctgct tgtttgttgccaaagttgga tgaattgaga 1681 gacgaaggta aggcttcttc cgctaagcaa agattgaagtgtgcttcctt gcaaaagttc 1741 ggtgaaagag ctttcaaggc ttgggctgtc gctagattgtctcaaagatt cccaaaggct 1801 gaattcgctg aagtttctaa gttggttact gacttgactaaggttcacac tgaatgttgt 1861 cacggtgact tgttggaatg tgctgatgac agagctgacttggctaagta catctgtgaa 1921 aaccaagact ctatctcttc caagttgaag gaatgttgtgaaaagccatt gttggaaaag 1981 tctcactgta ttgctgaagt tgaaaacgat gaaatgccagctgacttgcc atctttggct 2041 gctgacttcg ttgaatctaa ggacgtttgt aagaactacgctgaagctaa ggacgtcttc 2101 ttgggtatgt tcttgtacga atacgctaga agacacccagactactccgt tgtcttgttg 2161 ttgagattgg ctaagaccta cgaaactacc ttggaaaagtgttgtgctgc tgctgaccca 2221 cacgaatgtt acgctaaggt tttcgatgaa ttcaagccattggtcgaaga accacaaaac 2281 ttgatcaagc aaaactgtga attgttcgaa caattgggtgaatacaagtt ccaaaacgct 2341 ttgttggtta gatacactaa gaaggtccca caagtctccaccccaacttt ggttgaagtc 2401 tctagaaact tgggtaaggt cggttctaag tgttgtaagcacccagaagc taagagaatg 2461 ccatgtgctg aagattactt gtccgtcgtt ttgaaccaattgtgtgtttt gcacgaaaag 2521 accccagtct ctgatagagt caccaagtgt tgtactgaatctttggttaa cagaagacca 2581 tgtttctctg ctttggaagt cgacgaaact tacgttccaaaggaattcaa cgctgaaact 2641 ttcaccttcc acgctGATAT CTgtaccttg tccgaaaaggaaagacaaat taagaagcaa 2701 actgctttgg ttgaattggt caagcacaag ccaaaqgctactaaggaaca attgaaggct 2761 gtcatggatg atttcgctgc tttcgttgaa aagtgttgtaaggctgatga taaggaaact                                                   Bsu36I 2821tgtttcgctg aagaaggtaa gaagttggtc gctgcttccc aagctgCCTTAGGcttaggt >.....................rHA synth. gene......................>|>>>              BspEI 2881 ggttctggtggtTCCGGAgg tagtggtggc tccggtggtg aggcttgcaa tcttcctatCLinker---------------------------------->|--DX-890 (second coding)-->2941 gtccgtggcc cttgcatcgc cttttttcct cgttgggcct ttgacgccgt caaaggcaaa3001 tgcgtccttt ttccttacgg cggttgccag ggcaatggca ataaatttta tagcgagaaa3061 gagtgccgtg agtattgcgg cgtcccttaa taaGGTACCt aatAAGCTTa attcttatga----DX-890 (2nd coding)---->| 3121 tttatgattt ttattattaa ataagttataaaaaaaataa gtgtatacaa attttaaagt 3181 gactcttagg ttttaaaacg aaaattcttattcttgagta actctttcct gtaggtcagg                                                         SphI 3241ttgctttctc aggtatagca tgaggtcgct cttattgacc acacctctac cgGCATGCcg 3301agcaaatgcc tgcaaatcgc tccccatttc acccaattgt agatatgcta actccagcaa 3361tgagttgatg aatctcggtg tgtattttat gtcctcagag gacaacacct gttgtaatcg                 NotI 3421 ttcttccaca cggatcGCGG CCGC

The primary translation product of thisDPI-14-(GGS)₄GG-rHA-(GGS)₄GG-DX-890 fusion is as follows. 1 MKWVFIVSILFLFSSAYSRS LDKREAVREV CSEQAETGPC IAFFPRWYFD 51 VTEGKCAPFF YGGCGGNRNNFDTEEYCMAV CGSAGGSGGS GGSGGSGGDA 101 HKSEVAHRFK DLGEENFKAL VLIAFAQYLQQCPFEDHVKL VNEVTEFAKT 151 CVADESAENC DKSLHTLFGD KLCTVATLRE TYGEMADCCAKQEPERNECF 201 LQHKDDNPNL PRLVRPEVDV MCTAFHDNEE TFLKKYLYEI ARREPYFYAP251 ELLFFAKRYK AAFTECCQAA DKAACLLPKL DELRDEGKAS SAKQRLKCAS 301LQKFGERAFK AWAVARLSQR FPKAEFAEVS KLVTDLTKVH TECCHGDLLE 351 CADDRADLAKYICENQDSIS SKLKECCEKP LLEKSHCIAE VENDEMPADL 401 PSLAADFVES KDVCKNYAEAKDVFLGMFLY EYARRHPDYS VVLLLRLAKT 451 YETTLEKCCA AADPHECYAK VFDEFKPLVEEPQNLIKQNC ELFEQLGEYK 501 FQNALLVRYT KKVPQVSTPT LVEVSRNLGK VGSKCCKHPEAKRMPCAEDY 551 LSVVLNQLCV LHEKTPVSDR VTKCCTESLV NRRPCFSALE VDETYVPKEF601 NAETFTFHAD ICTLSEKERQ IKKQTALVEL VKHKPKATKE QLKAVMDDFA 651AFVEKCCKAD DKETCFAEEG KKLVAASQAA LGLGGSGGSG GSGGSGGEAC 701 NLPIVRGPCIAFFPRWAFDA VKGKCVLFPY GGCQGNGNKF YSEKECREYC 751 GVP

But as the first 24 amino acids constitute the fusion leader sequence,as described herein, the amino acid sequence of the secreted product areas follows: 1 EAVREVCSEQ AETGPCIAFF PRWYFDVTEG KCAPFFYGGC GGNRNNFDTE 51EYCMAVCGSA GGSGGSGGSG GSGGDAHKSE VAHRFKDLGE ENFKALVLIA 101 FAQYLQQCPFEDHVKLVNEV TEFAKTCVAD ESAENCDKSL HTLFGDKLCT 151 VATLRETYGE MADCCAKQEPERNECFLQHK DDNPNLPRLV RPEVDVMCTA 201 FHDNEETFLK KYLYEIARRH PYFYAPELLFFAKRYKAAFT ECCQAADKAA 251 CLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAVARLSQRFPKA 301 EFAEVSKLVT DLTKVHTECC HGDLLECADD RADLAKYICE NQDSISSKLK351 ECCEKPLLEK SHCIAEVEND EMPADLPSLA ADFVESKDVC KNYAEAKDVF 401LGMFLYEYAR RHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVFDE 451 FKPLVEEPQNLIKONCELFE QLGEYKFQNA LLVRYTKKVP QVSTPTLVEV 501 SRNLGKVGSK CCKHPEAKRMPCAEDYLSVV LNQLCVLHEK TPVSDRVTKC 551 CTESLVNRRP CFSALEVDET YVPKEFNAETFTFHADICTL SEKERQIKKQ 601 TALVELVKHK PKATKEQLKA VMDDFAAFVE KCCKADDKETCFAEEGKKLV 651 AASQAALGLG GSGGSGGSGG SGGEACNLPI VRGPCIAFFP RWAFDAVKGK701 CVLFPYGGCQ GNGNKFYSEK ECREYCGVP

Example 23 Amino-Acid Sequence of a DPI-14-(GGS)₄GG-HSA Fusion Protein

Table 33 shows the amino-acid sequence of a fusion of DPI14 via a linkercomprising (GGS)₄GG to HSA. Construction of a gene to encode the givensequence is simple using the methods and vectors described herein.DPI-14 is a potent inhibitor of HNE and the fusion to HSA produces amolecule with longer serum residence time.

Tables

TABLE 1 Amino-acid sequencer of Mature HSA from GenBank entry AAN17825(SEQ ID NO: 18) DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFAKTCVADESAE NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNPNLPRLVRPEV DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQAADKAACLLP KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAEVSKLVTDLTK VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCIAEVENDEMPA DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLAKTYKTTLEKC CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVRYTKKVPQVST PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVSDRVTKCCTES LVNRRPCFSA LEVDETYVPK EPNAETFTFH ADICTLSEKE RQIKKQTALVELVKHKPKAT KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASR AALGL

TABLE 2 Amino-acid sequences of DX-1000 and DX-88 DX-1000 (SEQ IDNO:_(——)) EAMHSFCAFKAETGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESL EECKKMCTRDDX-88 (SEQ ID NO:_(——))EAMHSFCAFKADDGPCRAAHPRWFFNIFTRQCEEFIYGGCEGNQNRFESL EECKKMCTRD

Table 5 DNA sequence of the N-terminal BglII-BamHI DPI-14 cDNA (SEQ IDNO:_(——)) AGATCTTTGGATAAGAGAGAAGCTGTTAGAGAAGTTTGTTCTGAACAAGCTGAAACTGGTCCATGTATTGCTTTCTTCCCAAGATGGTACTTCGATGTTACTGAAGGTAAGTGCGCGCCATTCTTCTACGGTGGTTGTGGTGGTAACAGAAACAACTTCGATACTGAAGAATACTGTATGGCTGTTTGTGGTTCTGCTGG TGGATCC

TABLE 6 DNA sequence of the C-terminal BamHI-HindIII DPI-14 cDNA (SEQ IDNO:_(——)) GGATCCGGTGGTGAAGCTGTTAGAGAAGTTTGTTCTGAACAAGCTGAAACTGGTCCATGTATTGCTTTCTTCCCAAGATGGTACTTCGATGTTACTGAAGGTAAGTGCGCGCCATTCTTCTACGGTGGTTGTGGTGGTAACAGAAACAACTTCGATACTGAAGAATACTGTATGGCTGTTTGTGGTTCTGCTTAAT AAGCTT

TABLE 7 DNA sequence of the N-terminal DPI-14-(GGS)₄GG-albumin fusioncoding region (SEQ ID NO:_(——))GAAGCTGTTAGAGAAGTTTGTTCTGAACAAGCTGAAACTGGTCCATGTATTGCTTTCTTCCCAAGATGGTACTTCGATGTTACTGAAGGTAAGTGCGCGCCATTCTTCTACGGTGGTTGTGGTGGTAACAGAAACAACTTCGATACTGAAGAATACTGTATGGCTGTTTGTGGTTCTGCTGGTGGATCCGGTGGTTCCGGTGGTTCTGGTGGTTCCGGTGGTGACGCTCACAAGTCCGAAGTCGCTCACCGGTTCAAGGACCTAGGTGAGGAAAACTTCAAGGCTTTGGTCTTGATCGCTTTCGCTCAATACTTGCAACAATGTCCATTCGAAGATCACGTCAAGTTGGTCAACGAAGTTACCGAATTCGCTAAGACTTGTGTTGCTGACGAATCTGCTGAAAACTGTGACAAGTCCTTGCACACCTTGTTCGGTGATAAGTTGTGTACTGTTGCTACCTTGAGAGAAACCTACGGTGAAATGGCTGACTGTTGTGCTAAGCAAGAACCAGAAAGAAACGAATGTTTCTTGCAACACAAGGACGACAACCCAAACTTGCCAAGATTGGTTAGACCAGAAGTTGACGTCATGTGTACTGCTTTCCACGACAACGAAGAAACCTTCTTGAAGAAGTACTTGTACGAAATTGCTAGAAGACACCCATACTTCTACGCTCCAGAATTGTTGTTCTTCGCTAAGAGATACAAGGCTGCTTTCACCGAATGTTGTCAAGCTGCTGATAAGGCTGCTTGTTTGTTGCCAAAGTTGGATGAATTGAGAGACGAAGGTAAGGCTTCTTCCGCTAAGCAAAGATTGAAGTGTGCTTCCTTGCAAAAGTTCGGTGAAAGAGCTTTCAAGGCTTGGGCTGTCGCTAGATTGTCTCAAAGATTCCCAAAGGCTGAATTCGCTGAAGTTTCTAAGTTGGTTACTGACTTGACTAAGGTTCACACTGAATGTTGTCACGGTGACTTGTTGGAATGTGCTGATGACAGAGCTGACTTGGCTAAGTACATCTGTGAAAACCAAGACTCTATCTCTTCCAAGTTGAAGGAATGTTGTGAAAAGCCATTGTTGGAAAAGTCTCACTGTATTGCTGAAGTTGAAAACGATGAAATGCCAGCTGACTTGCCATCTTTGGCTGCTGACTTCGTTGAATCTAAGGACGTTTGTAAGAACTACGCTGAAGCTAAGGACGTCTTCTTGGGTATGTTCTTGTACGAATACGCTAGAAGACACCCAGACTACTCCGTTGTCTTGTTGTTGAGATTGGCTAAGACCTACGAAACTACCTTGGAAAAGTGTTGTGCTGCTGCTGACCCACACGAATGTTACGCTAAGGTTTTCGATGAATTCAAGCCATTGGTCGAAGAACCACAAAACTTGATCAAGCAAAACTGTGAATTGTTCGAACAATTGGGTGAATACAAGTTCCAAAACGCTTTGTTGGTTAGATACACTAAGAAGGTCCCACAAGTCTCCACCCCAACTTTGGTTGAAGTCTCTAGAAACTTGGGTAAGGTCGGTTCTAAGTGTTGTAAGCACCCAGAAGCTAAGAGAATGCCATGTGCTGAAGATTACTTGTCCGTCGTTTTGAACCAATTGTGTGTTTTGCACGAAAAGACCCCAGTCTCTGATAGAGTCACCAAGTGTTGTACTGAATCTTTGGTTAACAGAAGACCATGTTTCTCTGCTTTGGAAGTCGACGAAACTTACGTTCCAAAGGAATTCAACGCTGAAACTTTCACCTTCCACGCTGATATCTGTACCTTGTCCGAAAAGGAAAGACAAATTAAGAAGCAAACTGCTTTGGTTGAATTGGTCAAGCACAAGCCAAAGGCTACTAAGGAACAATTGAAGGCTGTCATGGATGATTTCGCTGCTTTCGTTGAAAAGTGTTGTAAGGCTGATGATAAGGAAACTTGTTTCGCTGAAGAAGGTAAGAAGTTGGTCGCTGCTTCCCAAGCTGCTTTGGGTTTG

TABLE 8 Amino acid sequence of the N-terminal DPI-14-(GGS)₄GG-albuminfusion protein (SEQ ID NO:_(——))EAVREVCSEQAETGPCIAFFPRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSAGGSGGSGGSGGSGGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL

TABLE 9 DNA sequence of the C-terminal albumin- (GGS)₄GG-DPI-14 fusioncoding region (SEQ ID NO:_(——))GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTCAACATCATCTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCAAACGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAACCATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTTCTAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTAGGTGGTTCTGGTGGTTCCGGTGGTTCTGGTGGATCCGGTGGTGAAGCTGTTAGAGAAGTTTGTTCTGAACAAGCTGAAACTGGTCCATGTATTGCTTTCTTCCCAAGATGGTACTTCGATGTTACTGAAGGTAAGTGCGCGCCATTCTTCTACGGTGGTTGTGGTGGTAACAGAAACAACTTCGATACTGAAGAATACTGTATGGCTGTTTGTGGTTCTGCT

TABLE 10 Amino acid sequence of the C-terminal albumin-(GGS)₄GG-DPI-14fusion protein (SEQ ID NO:_(——))DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKABFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKNKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGSGGSGGSGGSGGEAVREVCSEQAETGPCIAFFPRWYFDVTEGKCAPFFYGGCGGNRNNFDTEE YCMAVCGSA

TABLE 11 DNA sequence of the C-terminal BamHI-HindIII DX-1000 cDNA (SEQID NO:_(——)) GGA TCC GGT GGT gag gct atg cat tcc ttc tgc gcc ttc aag gctgag act ggt cct tgt aga gct agg ttc gac cgt tgg ttc ttc aac atc ttc acgcgt cag tgc gag gaa ttc att tac ggt ggt tgt gaa ggt aac cag aac cgg ttcgaa tct cta gag gaa tgt aag aag atg tgc act cgt gac TAA TAA GCT T

TABLE 12 DNA sequence of the N-terminal BglII-BamHI-DX-890 cDNA (SEQ IDNO:_(——)) AGATCTTTGGATAAGAGAGAAGCCTGTAACTTGCCAATTGTTAGAGGTCCATGTATTGCTTTCTTCCCAAGATGGGCTTTCGATGCTGTTAAGGGTAAGTGTGTTTTGTTCCCATATGGTGGTTGTCAAGGTAACGGTAACAAGTTCTACTCTGAAAAGGAATGTAGAGAATACTGTGGTGTTCCAGGTGGATCC

TABLE 13 DNA sequence of the C-terminal BamHI-HindIII DX-890 cDNA (SEQID NO:_(——)) GGATCCGGTGGTGAAGCCTGTAACTTGCCAATTGTTAGAGGTCCATGTATTGCTTTCTTCCCAAGATGGGCTTTCGATGCTGTTAAGGGTAAGTGTGTTTTGTTCCCATATGGTGGTTGTCAAGGTAACGGTAACAAGTTCTACTCTGAAAAGGAATGTAQAGAATACTGTGGTGTTCCATAATAAGCTT

TABLE 14 DNA sequence of the N-terminal DX-890-(GGS)₄GG-albumin fusioncoding region (SEQ ID NO:_(——))GAAGCCTGTAACTTGCCAATTGTTAGAGGTCCATGTATTGCTTTCTTCCCAAGATGGGCTTTCGATGCTGTTAAGGGTAAGTGTGTTTTGTTCCCATATGGTGGTTGTCAAGGTAACGGTAACAAGTTCTACTCTGAAAAGGAATGTAGAGAATACTGTGGTGTTCCAGGTGGATCCGGTGGTTCCGGTGGTTCTGGTGGTTCCGGTGGTGACGCTCACAAGTCCGAAGTCGCTCACCGGTTCAAGGACCTAGGTGAGGAAAACTTCAAGGCTTTGGTCTTGATCGCTTTCGCTCAATACTTGCAACAATGTCCATTCGAAGATCACGTCAAGTTGGTCAACGAAGTTACCGAATTCGCTAAGACTTGTGTTGCTGACGAATCTGCTGAAAACTGTGACAAGTCCTTGCACACCTTGTTCGGTGATAAGTTGTGTACTGTTGCTACCTTGAGAGAAACCTACGGTGAAATGGCTGACTGTTGTGCTAAGCAAGAACCAGAAAGAAACGAATGTTTCTTGCAACACAAGGACGACAACCCAAACTTGCCAAGATTGGTTAGACCAGAAGTTGACGTCATGTGTACTGCTTTCCACGACAACGAAGAAACCTTCTTGAAGAAGTACTTGTACGAAATTGCTAGAAGACACCCATACTTCTACGCTCCAGAATTGTTGTTCTTCGCTAAGAGATACAAGGCTGCTTTCACCGAATGTTGTCAAGCTGCTGATAAGGCTGCTTGTTTGTTGCCAAAGTTGGATGAATTGAGAGACGAAGGTAAGGCTTCTTCCGCTAAGCAAAGATTGAAGTGTGCTTCCTTGCAAAAGTTCGGTGAAAGAGCTTTCAAGGCTTGGGCTGTCGCTAGATTGTCTCAAAGATTCCCAAAGGCTGAATTCGCTGAAGTTTCTAAGTTGGTTACTGACTTGACTAAGGTTCACACTGAATGTTGTCACGGTGACTTGTTGGAATGTGCTGATGACAGAGCTGACTTGGCTAAGTACATCTGTGAAAACCAAGACTCTATCTCTTCCAAGTTGAAGGAATGTTGTGAAAAGCCATTGTTGGAAAAGTCTCACTGTATTGCTGAAGTTGAAAACGATGAAATGCCAGCTGACTTGCCATCTTTGGCTGCTGACTTCGTTGAATCTAAGGACGTTTGTAAGAACTACGCTGAAGCTAAGGACGTCTTCTTGGGTATGTTCTTGTACGAATACGCTAGAAGACACCCAGACTACTCCGTTGTCTTGTTGTTGAGATTGGCTAAGACCTACGAAACTACCTTGGAAAAGTGTTGTGCTGCTGCTGACCCACACGAATGTTACGCTAAGGTTTTCGATGAATTCAAGCCATTGGTCGAAGAACCACAAAACTTGATCAAGCAAAACTGTGAATTGTTCGAACAATTGGGTGAATACAAGTTCCAAAACGCTTTGTTGGTTAGATACACTAAGAAGGTCCCACAAGTCTCCACCCCAACTTTGGTTGAAGTCTCTAGAAACTTGGGTAAGGTCGGTTCTAAGTGTTGTAAGCACCCAGAAGCTAAGAGAATGCCATGTGCTGAAGATTACTTGTCCGTCGTTTTGAACCAATTGTGTGTTTTGCACGAAAAGACCCCAGTCTCTGATAGAGTCACCAAGTGTTGTACTGAATCTTTGGTTAACAGAAGACCATGTTTCTCTGCTTTGGAAGTCGACGAAACTTACGTTCCAAAGGAATTCAACGCTGAAACTTTCACCTTCCACGCTGATATCTGTACCTTGTCCGAAAAGGAAAGACAAATTAAGAAGCAAACTGCTTTGGTTGAATTGGTCAAGCACAAGCCAAAGGCTACTAAGGAACAATTGAAGGCTGTCATGGATGATTTCGCTGCTTTCGTTGAAAAGTGTTGTAAGGCTGATGATAAGGAAACTTGTTTCGCTGAAGAAGTAAGAAGTTGGTCGCTGCTTCCCAAGCTGCTTTGGGTTTG

TABLE 15 Amino acid sequence of the N-terminal DX-890-(GGS)₄GG-albuminfusion protein (SEQ ID NO:_(——))EACNLPIVRGPCIAFFPRWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECREYCGVPGGSGGSGGSGGSGGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDVKLVNEVTEFSAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAGHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFKEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSKRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKIVAASQ AALGL

TABLE 16 DNA sequence of the C-terminal albumin-(GGS)₄GG-DX-890 fusioncoding region (SEQ ID NO:_(——)) GATGCACACA AGAGTGAGGT TGCTCATCGGTTTAAAGATT TGGGAGAAGA AAATTTCAAA GCCTTGGTGT TGATTGCCTT TGCTCAGTATCTTCAGCAGT GTCCATTTGA AGATCATGTA AAATTAGTGA ATGAAGTAAC TGAATTTGCAAAAACATGTG TTGCTGATGA GTCAGCTGAA AATTGTGACA AATCACTTCA TACCCTTTTTGGAGACAAAT TATGCACAGT TGCAACTCTT CGTGAAACCT ATGGTGAAAT GGCTGACTGCTGTGCAAAAC AAGAACCTGA GAGAAATGAA TGCTTCTTGC AACACAAAGA TGACAACCCAAACCTCCCCC GATTGGTGAG ACCAGAGGTT GATGTGATGT GCACTGCTTT TCATGACAATGAAGAGACAT TTTTGAAAAA ATACTTATAT GAAATTGCCA GAAGACATCC TTACTTTTATGCCCCGGAAC TCCTTTTCTT TGCTAAAAGG TATAAAGCTG CTTTTACAGA ATGTTGCCAAGCTGCTGATA AAGCTGCCTG CCTGTTGCCA AAGCTCGATG AACTTCGGGA TGAAGGGAAGGCTTCGTCTG CCAAACAGAG ACTCAAGTGT GCCAGTCTCC AAAAATTTGG AGAAAGAGCTTTCAAAGCAT GGGCAGTAGC TCGCCTGAGC CAGAGATTTC CCAAAGCTGA GTTTGCAGAAGTTTCCAAGT TAGTGACAGA TCTTACCAAA GTCCACACGG AATGCTGCCA TGOAGATCTGCTTGAATGTG CTGATGACAG GGCGGACCTT GCCAAGTATA TCTGTGAAAA TCAAGATTCGATCTCCAGTA AACTGAAGGA ATGCTGTGAA AAACCTCTGT TGGAAAAATC CCACTGCATTGCCGAAGTGG AAAATGATGA GATGCCTGCT GACTTGCCTT CATTAGCTGC TGATTTTGTTGAAAGTAAGG ATGTTTGCAA AAACTATGCT GAGGCAAAGG ATGTCTTCCT GGGCATGTTTTTGTATGAAT ATGCAAGAAG GCATCCTGAT TACTCTGTCG TGCTGCTGCT GAGACTTGCCAAGACATATG AAACCACTCT AGAGAAGTGC TGTGCCGCTG CAGATCCTCA TGAATGCTATGCCAAAGTGT TCGATGAATT TAAACCTCTT GTGGAAGAGC CTCAGAATTT AATCAAACAAAATTGTGAGC TTTTTGAGCA GCTTGGAGAG TACAAATTCC AGAATGCGCT ATTAGTTCGTTACACCAAGA AAGTACCCCA AGTGTCAACT CCAACTCTTG TAGAGGTCTC AAGAAACCTAGGAAAAGTGG GCAGCAAATG TTGTAAACAT CCTGAAGCAA AAAGAATGCC CTGTGCAGAAGACTATCTAT CCGTGGTCCT GAACCAGTTA TGTGTGTTGC ATGAGAAAAC GCCAGTAAGTGACAGAGTCA CCAAATGCTG CACAGAATCC TTGGTGAACA GGCGACCATG CTTTTCAGCTCTGGAAGTCG ATGAAACATA CGTTCCCAAA GAGTTTAATG CTGAAACATT CACCTTCCATGCAGATATAT GCACACTTTC TGAGAAGGAG AGACAAATCA AGAAACAAAC TGCACTTGTTGAGCTCGTGA AACACAAGCC CAAGGCAACA AAAGAGCAAC TGAAAGCTGT TATGGATGATTTCGCAGCTT TTGTAGAGAA GTGCTGCAAG GCTGACGATA AGGAGACCTG CTTTGCCGAGGAGGGTAAAA AACTTGTTGC TGCAAGTCAA GCTGCCTTAG GCTTAGGTGG TTCTGGTGGTTCCGGTGGTT CTGGTGGATC CGGTGGTGAA GCCTGTAACT TGCCAATTGT TAGAGGTCCATGTATTGCTT TCTTCCCAAG ATGGGCTTTC GATGCTGTTA AGGGTAAGTG TGTTTTGTTCCCATATGGTG GTTGTCAAGG TAACGGTAAC AAGTTCTACT CTGAAAAGGA ATGTAGAGAATACTGTGGTG TTCCA TABLE 17 Amino acid sequence of the C-terminalalbumin-(GGS)₄GG-DX-890 fusion protein (SEQ ID NO:_(——)) DAHKSEVAHRFKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAE NCDKSLHTLFGDKLCTVATL RETYGEMADC CAKOEPERNE CFLQHKDDNP NLPRLVRPEV DVMCTAFHDNEETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP KLDELRDEGKASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKINTDLTK VHTECCHGDLLECADDRADL AKYICENODS ISSKLKECCE KPLLEKSHCI AEVENDEMPA DLPSLAADFVESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC CAAADPHECYAKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVST PTLVEVSRNLGKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES LVNRRPCFSALEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT KEQLKAVMDDFAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGLGGSGG SGGSGGSGGE ACNLPIVRGPCIAFFPRWAF DAVKGKCVLF PYGGCQGNGN KFYSEKECREY CGVP

TABLE 18 DNA sequence of the N-terminal BglII-BamHI DX-88 cDNA (SEQ IDNO:_(——)) AGA TCT TTG GAT AAG AGA GAA GCT ATG CAC TCT TTG TGT GGT TTCAAG GCT GAG GAG GGT CCG TGC AGA GCT GCT GAG CCA AGA TGG TTG TTG AAG ATCTTC AGG CGA CAA TGC GAG GAG TTG ATC TAG GGT GGT TGT GAG GGT AAG GAA AAGAGA TTC GAG TGT GTA GAG GAG TGT AAG AAG ATG TGT ACT AGA GAC GGT GGA TCC

TABLE 19 DNA sequence of the N-terminalDX-88-(GGS)_(4GG-albumin fusion coding region) (SEQ ID NO:_(——)) GAA GCTATG CAC TCT TTC TGT GCT TTC AAG GCT GAC GAC GGT CCG TGC AGA GCT GCT CACCCA AGA TGG TTC TTC AAC ATC TTC ACG CGA CAA TGC GAG GAG TTC ATC TAC GGTGGT TGT GAG GGT AAC CAA AAC AGA TTC GAG TCT CTA GAG GAG TGT AAG AAG ATGTGT ACT AGA GAC GGT GGATCCGGTGGTTCCGGTGGTTCTGGTGGTTCCGGTGGTGACGCTCACAAGTCCGAAGTCGCTCACCGGTTCAAGGACCTAGGTGAGGAAAACTTCAAGGCTTTGGTCTTGATCGCTTTCGCTCAATACTTGCAACAATGTCCATTCGAAGATCACGTCAAGTTGGTCAACGAAGTTACCGAATTCGCTAAGACTTGTGTTGCTGACGAATCTGCTGAAAACTGTGACAAGTCCTTGCACACCTTGTTCGGTGATAAGTTGTGTACTGTTGCTACCTTGAGAGAAACCTACGGTGAAATGGCTGACTGTTGTGCTAAGCAAGAACCAGAAAGAAACGAATGTTTCTTGCAACACAAGGACGACAACCCAAACTTGCCAAGATTGGTTAGACCAGAAGTTGACGTCATGTGTACTGCTTTCCACGACAACGAAGAAACCTTCTTGAAGAAGTACTTGTACGAAATTGCTAGAAGACACCCATACTTCTACGCTCCAGAATTGTTGTTCTTCGCTAAGAGATACAAGGCTGCTTTCACCGAATGTTGTCAAGCTGCTGATAAGGCTGCTTGTTTGTTGCCAAAGTTGGATGAATTGAGAGACGAAGGTAAGGCTTCTTCCGCTAAGCAAAGATTGAAGTGTGCTTCCTTGCAAAAGTTCGGTGAAAGAGCTTTCAAGGCTTGGGCTGTCGCTAGATTGTCTCAAAGATTCCCAAAGGCTGAATTCGCTGAAGTTTCTAAGTTGGTTACTGACTTGACTAAGGTTCACACTGAATGTTGTCACGGTGACTTGTTGGATCGTGCTGATGACAGAGCTGACTTGGCTAAGTACATCTGTGAAAACCAAGACTCTATCTCTTCCAAGTTGAAGGAATGTTGTGAAAAGCCATTGTTGGAAAAGTCTCACTGTATTGCTGAAGTTGAAAACGATGAAATGCCAGCTGACTTGCCATCTTTGGCTGCTGACTTCGTTGAATCTAAGGACGTTTGTAAGAACTACGCTGAAGCTAAGGACGTCTTCTTGGGTATGTTCTTGTACGAATACGCTAGAAGACACCCAGACTACTCCGTTGTCTTGTTGTTGAGATTGGCTAAGACCTACGAAACTACCTTGGAAAAGTGTTGTGCTGCTGCTGACCCACACGAATGTTACGCTAAGGTTTTCGATGAATTCAAGCCATTGGTCGAAGAACCACAAAACTTGATCAAGCAAAACTGTGAATTGTTCGAACAATTGGGTGAATACAAGTTCCAAAACGCTTTGTTGGTTAGATACACTAAGAAGGTCCCACAAGTCTCCACCCCAACTTTGGTTGAAGTCTCTAGAAACTTGGGTAAGGTCGGTTCTAAGTGTTGTAAGCACCCAGAAGCTAAGAGAATGCCATGTGCTGAAGATTACTTGTCCGTCGTTTTGAACCAATTGTGTGTTTTGCACGAAAAGACCCCAGTCTCTGATAGAGTCACCAAGTGTTGTACTGAATCTTTGGTTAACAGAAGACCATGTTTCTCTGCTTTGGAAGTCGACGAAACTTACGTTCCAAAGGAATTCAACGCTGAAACTTTCACCTTCCACGCTGATATCTGTACCTTGTCCGAAAAGGAAAGACAAATTAAGAAGCAAACTGCTTTGGTTGAATTGGTCAAGCACAAGCCAAAGGCTACTAAGGAACAATTGAAGGCTGTCATGGATGATTTCGCTGCTTTCGTTGAAAAGTGTTGTAAGGCTGATGATAAGGAAACTTGTTTCGCTGAAGAAGGTAAGAAGTTGGTCGCTGCTTCCCAAGCTGCTTTGGGTTTG

TABLE 20 AA sequence of DX-88::HSA (SEQ ID NO:_(——)) EAMHSFCAFKADDGPCRAAH PRWFFNIFTR QCEEFIYGGC EGNQNRFESL EECKKMCTRD GGSGGSGGSGGSGGDAHKSE VAHRFKDLGE ENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVADESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEP ERNECFLQHK DDNPNLPRLVRPEVDVMCTA FHDNEETFLK KYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAACLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAV ARLSQRFPKA EFAEVSKLVTDLTKVHTECC HGDLLECADD RADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVENDEMPADLPSLA ADFVESKDVC KNYAEAKDVF LGMFLYEYAR RHPDYSVVLL LRLAKTYETTLEKCCAAADP HECYAKVFDE FKPLVEEPQN LIKQNCELFE QLGEYKFQNA LLVRYTKKVPQVSTPTLVEV SRNLGKVGSK CCKHPEAKRM PCAEDYLSVV LNQLCVLHEK TPVSDRVTKCCTESLVNRRP CFSALEVDET YVPKEFNAET FTFHADICTL SEKERQIKKQ TALVELVKHKPKATKEH

TABLE 21 DNA sequence of the C-terminal BamHI-HindIII DX-88 cDNA (SEQ IDNO:_(——)) GGA TCC GGT GOT GAA GCT ATG CAC TCT TTC TGT GCT TTC AAG GCTGAC GAC GGT CCG TGC AGA GCT GCT CAC CCA AGA TGG TTC TTC AAC ATC TTC ACGCGA CAA TGC GAG GAG TTC ATC TAC GGT GGT TGT GAG GGT AAC CAA AAC AGA TTCGAG TCT CTA GAG GAG TGT AAG AAG ATG TGT ACT AGA GAC TAA TAA GCT T

TABLE 22 HSA::(GGS)4GG::DX-88 (SEQ ID NO: ) gat gca cac aag agt gag gttgct cat cgg ttt aaa gat ttg gga gaa gaa aat ttc aaa gcc ttg gtg ttg attgcc ttt gct cag tat ctt cag cag tgt cca ttt gaa gat cat gta aaa tta gtgaat gaa gta act gaa ttt gca aaa aca tgt gtt gct gat gag tca gct gaa aattgt gac aaa tca ctt cat acc ctt ttt gga gac aaa tta tgc aca gtt gca actctt cgt gaa acc tat ggt gaa atg gct gac tgc tgt gca aaa caa gaa cct gagaga aat gaa tgc ttc ttg caa cac aaa gat gac aac cca aac ctc ccc cga ttggtg aga cca gag gtt gat gtg atg tgc act gct ttt cat gac aat gaa gag acattt ttg aaa aaa tac tta tat gaa att gcc aga aga cat cct tac ttt tat gccccg gaa ctc ctt ttc ttt gct aaa agg tat aaa gct gct ttt aca gaa tgt tgccaa gct gct gat aaa gct gcc tgc ctg ttg cca aag ctc gat gaa ctt cgg gatgaa ggg aag gct tcg tct gcc aaa cag aga ctc aag tgt gcc agt ctc caa aaattt gga gaa aga gct ttc aaa gca tgg gca gta gct cgc ctg agc cag aga tttccc aaa gct gag ttt gca gaa gtt tcc aag tta gtg aca gat ctt acc aaa gtccac acg gaa tgc tgc cat gga gat ctg ctt gaa tgt gct gat gac agg gcg gacctt gcc aag tat atc tgt gaa aat caa gat tcg atc tcc agt aaa ctg aag gaatgc tgt gaa aaa cct ctg ttg gaa aaa tcc cac tgc att gcc gaa gtg gaa aatgat gag atg cct gct gac ttg cct tca tta gct gct gat ttt gtt gaa agt aaggat gtt tgc aaa aac tat gct gag gca aag gat gtc ttc ctg ggc atg ttt ttgtat gaa tat gca aga agg cat cct gat tac tct gtc gtg ctg ctg ctg aga cttgcc aag aca tat gaa acc act cta gag aag tgc tgt gcc gct gca gat cct catgaa tgc tat gcc aaa gtg ttc gat gaa ttt aaa cct ctt gtg gaa gag cct cagaat tta atc aaa caa aat tgt gag ctt ttt gag cag ctt gga gag tac aaa ttccag aat gcg cta tta gtt cgt tac acc aag aaa gta ccc caa gtg tca act ccaact ctt gta gag gtc tca aga aac cta gga aaa gtg ggc agc aaa tgt tgt aaacat cct gaa gca aaa aga atg ccc tgt gca gaa gac tat cta tcc gtg gtc ctgaac cag tta tgt gtg ttg cat gag aaa acg cca gta agt gac aga gtc acc aaatgc tgc aca gaa tcc ttg gtg aac agg cga cca tgc ttt tca gct ctg gaa gtcgat gaa aca tac gtt ccc aaa gag ttt aat gct gaa aca ttc acc ttc cat gcagat ata tgc aca ctt tct gag aag gag aga caa atc aag aaa caa act gca cttgtt gag ctc gtg aaa cac aag ccc aag gca aca aaa gag caa ctg aaa gct gttatg gat gat ttc gca gct ttt gta gag aag tgc tgc aag gct gac gat aag gagacc tgc ttt gcc gag gag ggt aaa aaa ctt gtt gct gca agt caa gct gcc ttaggc tta ggt ggt tct ggt ggt tcc ggt ggt tct ggt gga tcc ggt ggt GAA GCTATG CAC TCT TTC TGT GCT TTC AAG GCT GAC GAC GGT CCG TGC AGA GCT GCT CACCCA AGA TGG TTC TTC AAC ATC TTC ACG CGA CAA TGC GAG GAG TTC ATC TAC GGTGGT TGT GAG GGT AAC CAA AAC AGA TTC GAG TCT CTA GAG GAG TGT AAG AAG ATGTGT ACT AGA GAC

TABLE 23 AA sequence of mature protein encoded in Table 22 (SEQ. IDNO:_(——)) DAHKSEVAHRFKDLGEENFKALVLIAFAQY LQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADC CAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERA FKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDS ISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMF LYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQ NCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAE DYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFH ADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAE EGKKLVAASQAALGLGGSGGSGGSGGSGGEAMHSFCAFKADDGPCRAAHPRWFFNIFTRQ CEEFIYGGCEGNQNRFESLEECKKMCTRD

TABLE 25 NotI cassette of pDB2300X1 with 2xGS linkers (SEQ ID NO:_(——))1 GCGGCCGCcc gtaatgcggt atcgtgaaag cgaaaaaaaa actaacagta gataagacagNotI.... 61 atagacagat agagatggac gagaaacagg gggggagaaa aggggaaaagagaaggaaag 121 aaagactcat ctatcgcaga taagacaatc aaccctcatG GCGCCtccaaccaccatccg                                           NarI... 181cactagggac caAGCGCTcg caccgttagc aacgcttgac tcacaaacca actGCCGGCt AfeI..    NgoMIV 241 gaaagagctt gtgcaatggg agtgccaatt caaaggagccgaatacgtct gctcgccttt 301 taagaggctt tttgaacact gcattgcacc cgacaaatcagccactaact acgaggtcac 361 ggacacatat accaatagtt aaaaattaca tatactctatatagcacagt agtgtgataa 421 ataaaaaatt ttgccaagac ttttttaaaC TGCACccgacagatcaggtc tgtgcctact                                BsgI... 481atgcacttat gcccggggtc ccgggaggag aaaaaacgag ggctgggaaa tgtccgtgga 541ctttaaacgc tccgggttag cagagtaGCA gggcttTCGg ctttggaaat ttaggtgact                             BcgI......... 601 tgttgaaaaa gcaaaatttgggctcagtaa tgCCActgca gTGGcttatc acgccaggac                                   BstXI                                      PStI... 661 tgcgggagtg gcgggggcaaacacacccgc gataaagagc gcgatgaata taaaaggggg 721 ccaatgttac gtcccgttatattggagttc ttcccataca aaCTTAAGag tccaattagc                                              AflII. 781 ttcatcgccaataaaaaaac AAGCTTaacc taattctaac aagcaaag                       HindIII(1/2)   1   2   3   4   5   6   7   8   9   10  11  12  13  14  15    M   K   W   V   F   I   V   S   I   L   F   L   F   S   S 829 atg aagtgg gtt ttc atc gtc tcc att ttg ttc ttg ttc tcc tct 16  17  18  19  20  21  22  23  24  25  26  27  28  29  30 A   Y   S   R   S   L   D   K   R   G   G   S   G   G   S 874 gct tactct AGA TCT ttg gat aag aga ggt GGA TCC ggt ggt tcc            BglII..                        BamHI.. 31  32  33  34  35  36  37  38  39  40  41  42  43  44  45 G  G  S  G  G  S  G  G  D  A  H  K  S  E  V 919 ggt ggt tct ggt ggt tccggt ggt gac gct cac aag tcc gaa gtc 46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 A  H  R  F  K  D  L  G  E  E  N  F  K  A  L 964 gct cAC CCG Ttc aag gaCCTA GGt gag gaa aac ttc aag gct ttg      AgeI....         AvrII... 61  62  63  64  65  66  67  68  69  70  71  72  73  74  75 V  L  I  A  F  A  Q  Y  L  Q  Q  C  P  F  E 1009 gtc ttg atc gct ttcgct caa tac ttg caa caa tgt cca ttC gaa 76  7  78  79  80  81  82  83  84  85  86  87  88  89  90 D  H  V  K  L  V  N  E  V  T  E  F  A  K  T 1054 gat CAC GTC aag ttggtc aac gaa gtt acc gaa ttc gct aag act     BmgBI.. 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 C  V  D  E  S  A  E  N  C  D  K  S  L  H 1099 tgt gtt gct gac gaa tctgct gaa aac tgt gac aag tcc ttg cac 106 107 108 109 110 111 112 113 114115 116 117 118 119 120 T   L   F   G   D   K   L   C   T   V   A   T   L   R   E 1144 acc ttgttc ggt gat aag ttg tgt act gtt gct acc ttg aga gaa 121 122 123 124 125126 127 128 129 130 131 132 133 134 135 T   Y   C   E   M   A   D   C   C   A   K   Q   E   P   E 1189 acc tacggt gaa atg gct gac tgt tgt gct aag caa gaa cca gaa 136 137 138 139 140141 142 143 144 145 146 147 148 149 150 R   N   E   C   F   L   Q   H   K   D   D   N   P   N   L 1234 aga aacgaa tgt ttc ttg caa cac aag gac gac aac cca aac ttg 151 152 153 154 155156 157 158 159 160 161 162 163 164 165 P   R   L   V   R   P   E   V   D   V   M   C   T   A   F 1279 cca agattg gtt aga cca gaa gtt gac gtc atg tgt act gct ttc 166 167 168 169 170171 172 173 174 175 176 177 178 179 180 H   D   N   E   E   T   F   L   K   K   Y   L   Y   E   I 1324 cac gacaac gaa gaa acc ttc ttg aag aAG TAC Ttg tac gaa att                                     ScaI.... 181 182 183 184 185 186187 188 189 190 191 192 193 194 195 A   R   R   H   P   Y   F   Y   A   P   B   L   L   F   F 1369 gct agaaga cac cca tac ttc tac gct cca gaa ttg ttg ttC ttC 196 197 198 199 200201 202 203 204 205 206 207 208 209 210 A   K   R   Y   K   A   A   F   T   E   C   C   Q   A   A 1414 gct aagaga tac aag gct gCt ttc acc gaa tgt tgt caa gct gct 211 212 213 214 215216 217 218 219 220 221 222 223 224 225 D   K   A   A   C   L   L   P   K   L   D   E   L   R   D 1459 gat aaggct gct tgt ttg ttg cca aag ttg gat gaa ttg aga gac 226 227 228 229 230231 232 233 234 235 236 237 238 239 240 E   G   K   A   S   S   A   K   Q   R   L   K   C   A   S 1504 gaa ggtaag gct tct tcc gct aag caa aga ttg aag tgt gct tcc 241 242 243 244 245246 247 248 249 250 251 252 253 254 255 L   Q   K   F   G   E   R   A   F   K   A   W   A   V   A 1549 ttg caaaag ttc ggt gaa aga gct ttc aag gct tgg gct gtc gct 256 257 258 259 260261 262 263 264 265 266 267 268 269 270 R   L   S   Q   R   F   P   K   A   E   F   A   E   V   S 1594 aga ttgtct caa aga ttc ccaaag gct gaa ttc gCt gaa gtt tct 271 272 273 274 275276 277 278 279 280 281 282 283 284 285 K   L   V   T   D   L   T   K   V   H   T   E   C   C   H 1639 aag ttggtt act gac ttg act aag gtt cac act gaa tgt tgt cac 286 287 288 289 290291 292 293 294 295 296 297 298 299 300 G   D   L   L   E   C   A   D   D   R   A   D   L   A   K 1684 ggt gacttg ttg gaa tgt gct gat gac aga gct gac ttg gct aag 301 302 303 304 305306 307 308 309 310 311 312 313 314 315 Y   I   C   E   N   Q   D   S   I   S   S   K   L   K   E 1729 tac atctgt gaa aac caa gac tct atC TCT TCc aag ttg aag gaa                                         EarI.... 316 317 318 319 320321 322 323 324 325 326 327 328 329 330 C   C   S   K   P   L   L   E   K   S   H   C   I   A   E 1774 tgt tgtgaa aag cca ttg ttg gaa aag tct cac tgt att gct gaa 331 332 333 334 335336 337 338 339 340 341 342 343 344 345 V   E   N   D   E   M   P   A   D   L   P   S   L   A   A 1819 gtt gaaaac gat gaa atg cCA GCT Gac ttg cca tct ttg gct gct                         PvuII... 346 347 348 349 350 351 352 353 354355 356 357 358 359 360 D   F   V   E   S   K   D   V   C   K   N   Y   A   E   A 1864 gac ttcgtt gaa tct aag gac gtt tgt aag aac tac gct gaa gct 361 362 363 364 365366 367 368 369 370 371 372 373 374 375 K   D   V   F   L   G   M   F   L   Y   E   Y   A   R   R 1909 aag gacgtc ttc ttg ggt atg ttc ttg tac gaa tac gct aga aga 376 377 378 379 380381 382 383 384 385 386 387 388 389 390 H   P   D   Y   S   V   V   L   L   L   R   L   A   K   T 1954 cac ccagac tac tcc gtt gtc ttg ttg ttg aga ttg gct aag acc 391 392 393 394 395396 397 398 399 400 401 402 403 404 405 Y   E   T   T   L   E   K   C   C   A   A   A   D   P   H 1999 tac gaaact acc ttg gaa aag tgt tgt gct gct gct gac cca cac 406 407 408 409 410411 412 413 414 415 416 417 418 419 420 E   C   Y   A   K   V   F   D   E   F   K   P   L   V   E 2044 gaa tgttac gct aag gtt ttc gat gaa ttc aag cca ttg gtc gaa 421 422 423 424 425426 427 428 429 430 431 432 433 434 435 E   P   Q   N   L   I   K   Q   N   C   E   L   F   E   Q 2089 gaa ccacaa aac tTG ATC Aag caa aac tgt gaa ttg ttc gaa caa                 BclI.... 436 437 438 439 440 441 442 443 444 445 446447 448 449 450 L   G   E   Y   K   F   Q   N   A   L   L   V   R   Y   T 2134 ttg ggtgaa tac aag ttc caa aac gct ttg ttg gtt aga tac act 451 452 453 454 455456 457 458 459 460 461 462 463 464 465 K   K   V   P   Q   V   S   T   P   T   L   V   E   V   S 2179 aag aaggtc cca caa gtc tCC Acc cca act tTG Gtt gaa gtc TCT                         XcmI................ 466 467 468 469 470 471472 473 474 475 476 477 478 479 480 R   N   L   G   K   V   G   S   K   C   C   K   H   P   E 2224 AGA aacttg ggt aag gtc ggt tct aag tgt tgt aag cac cca gaa 481 482 483 484 485486 487 488 489 490 491 492 493 494 495 A   K   R   M   P   C   A   E   D   Y   L   S   V   V   L 2269 gct aagaGA ATG Cca tgt gct gaa gat tac ttg tcc gtc gtt ttg          BsmI....496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 N   Q   L   C   V   L   H   E   K   T   P   V   S   D   R 2314 aac caattg tgt gtt ttg cac gaa aaG ACc cca GTC tct gat aga                                  PshAI........                                          AlwNI....... 511 512 513 514515 516 517 518 519 520 521 522 523 524 525 V   T   K   C   C   T   E   S   L   V   N   R   R   P   C 2359 gtC ACcaaG TGt tgt act gaa tct ttg GTT AAC aga aga cca tgt  DraIII......                      HpaI... 526 527 528 529 530 531 532533 534 535 536 537 538 539 540 F   S   A   L   E   V   D   E   T   Y   V   P   K   B   F 2404 ttc tctgct ttg gaa GTC GAC gaa act tac gtt cca aag GAA TTC                    SalI... 541 542 543 544 545 546 547 548 549 550 551552 553 554 555 N   A   E   T   F   T   F   H   A   D   I   C   T   L   S 2449 aac gctgaa act ttc acc ttc cac gct GAT ATC tgt acc ttg tcc                                    EcoRV.. 556 557 558 559 560 561 562563 564 565 566 567 568 569 570 E   K   E   R   Q   I   K   K   Q   T   A   L   V   E   L 2494 gaa aaggaa aga caa att aag aag caa act gct ttg gtt gaa ttg 571 572 573 574 575576 577 578 579 580 581 582 583 584 585 V   K   H   K   P   K   A   T   K   E   Q   L   K   A   V 2539 gtc aagcac aag cca aag gct act aag gaa caa ttg aag gct gtc 586 587 588 589 590591 592 593 594 595 596 597 598 599 600 M   D   D   F   A   A   F   V   E   K   C   C   K   A   D 2584 atg gatgat ttc gct gct ttc gtt gaa aag tgt tgt aag gct gat 601 602 603 604 605606 607 608 609 610 611 612 613 614 615 D   K   E   T   C   F   A   E   E   G   K   K   L   V   A 2629 gat aaggaa act tgt ttc gct gaa gaa ggt aag aag ttg gtc gct 616 617 618 619 620621 622 623 624 625 626 627 628 629 630 A   S   Q   A   A   L   G   L   G   G   S   G   G   S   G 2674 gct tcccaa gct gCC TTA GGc tta ggt ggt tct ggt ggt tcc ggt                 Bsu36I... 631 632 633 634 635 636 637 638 G   S   G   G   S   G   G   T 2719 ggt TCC GGA ggt tcc ggt GGTACC       taa tAA GCTTa attcttatga    BspEI..             KpnI...       Stop Stop                                           HindlIl (2/2) 2764 tttatgatttttattattaa ataagTTATA Aaaaaaataa gtGTATACaa attttaaagt                           PsiI...            BstZ17I 2824 gactcttaggttttaaaacg aaaattctta ttcttgagta agtctttcct gtaggtcagg 2884 ttgctttctcaggtatagca tgaggtcgct cttattgacc acacctctac cgGCATGCcg                                                        SphI.. 2944agcaaatgcc tgcaaatcgc tccccatttc acccaattgt agatatgcta actcc3004tgagttgatg aatctcggtg tgtattttat gtcctcagag gacaacacCt gttgtaatcg 3064ttcttccaca cggatCGCGG CCGC                 NotI

TABLE 26 NotI cassette of pDB2300X2 with DX890 (Nterm) and Cterm linkerready for second DX890 (SEQ. ID NO:_(——)) 1 GCGGCCGCcc gtaatgcggtatcgtgaaag cgaaaaaaaa actaacagta gataagacag NotI.... 61 atagacagatagagatggac gagaaacagg gggggagaaa aggggaaaag agaaggaaag 121 aaagactcatctatcgcaga taagacaatc aaccctcatG GCGCCtccaa ccaccatccg                                          Nar... 181 cactagggaccaAGCGCTcg caccgttagc aacgcttgac tcacaaacca actGCCGGCt             AfeI..                                       NgoMIV 241gaaagagctt gtgcaatggg agtgccaatt caaaggagcc gaatacgtct gctcgccttt 301taagaggctt tttgaacact gcattgcacc cgacaaatca gccactaact acgaggtcac 361ggacacatat accaatagtt aaaaattaca tatactctat atagcacagt agtgtgataa 421ataaaaaatt ttgccaagac ttttttaaaC TGCACccgac agatcaggtc tgtgcctact                               BsgI... 481 atgcacttat gcccggggtcccgggaggag aaaaaacgag ggctgggaaa tgtccgtgga 541 ctttaaacgc tccgggttagcagagtaGCA gggcttTCGg ctttggaaat ttaggtgact                             BcgI......... 601 tgttgaaaaa gcaaaatttgggctcagtaa tgCCActgca gTGGcttatc acgccaggac                                   BstXI                                      PStI... 661 tgcgggagtg gcgggggcaaacacacccgc gataaagagc gcgatgaata taaaaggggg 721 ccaatgttac gtcccgttatattggagttc ttcccataca aaCTTAAGag tccaattagC                                              AflII. 781 ttcatcgccaataaaaaaac AAGCTTaacc taattctaac aagcaaag                       HindIII(1/2)  Signal sequence--------------------------------------------   1   2   3   4   5   6   7   8   9   10   11   12   13   14   15  M   K   W   V   F   I   V   S   I   L   F   L   F   S   S 829  atg aagtgg gtt ttc atc gtc tcc att ttg ttc ttg ttc tcc tct  Signalsequence------------------->  DX-890----------------  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  A   Y   S   R   S   L   D   K   R   E   A   C   N   L   P 874  gct tactct AGA TCT ttg gat aag aga gaa gcc tgt aac ttg cca                BglII..          XbaI...(1/2)  DX890continued--------------------------------------------  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  I   V   R   G   P   C   I   A   F   F   P   R   W   A   F 919  att gttaga ggt cca tgt att gct ttc ttc cca aga tgg gct ttc  DX890continued--------------------------------------------  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  D   A   V   K   G   K   C   V   L   F   P   Y   G   G   C 964  gat gctgtt aag ggt aag tgt gtt ttg ttc CCA tat ggT GGt tgt                                         PflMI.........                                          NdeI....  DX890continued--------------------------------------------  61  62  63  64  65  66  67  68  69  70  71  72  73  74  75  Q   G   N   G   N   K   F   Y   S   E   K   E   C   R   S 1009  caaggt aac ggt aac aag ttc tac tct gaa aag gaa tgt aga gaa  DX890continued--->  Linker--------------------------------  76  77  78  79  80  81  82  83  84  85  86  87  88  89  90  Y   C   G   V   P   G   G   S   G   C   S   C   G   S   C 1054  tactgt ggt gtt cca ggt GGA TCC ggt ggt tcc ggt ggt tct ggt                         BamHI..   Linker------->  rHA--------------> toresidue 679 91  92  93  94  95  96  97  98  99  100  101  102  103  104  105  G   S   C   G   D   A   H   K   S   S   V   A   H   R   F 1099  ggttcc ggt ggt gac gct cac aag tcc gaa gtc gct cAC CGG Ttc                                                   AgeI....  106 107 108109 110 111 112 113 114 115 116 117 118 119 120  K   D   L   G   S   E   N   F   K   A   L   V   L   I   A 1144  aaggaC CTA GGt gag gaa aac ttc aag gct ttg gtc ttg atc gct        AvrII... 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135  F   A   Q   Y   L   Q   Q   C   P   F   E   D   H   V   K 1189  ttcgct caa tac ttg caa caa tgt cca ttc gaa gat CAC GTC aag                                                   BmgBI..  136 137 138139 140 141 142 143 144 145 146 147 148 149 150  L   V   N   E   V   T   E   F   A   K   T   C   V   A   D 1234  ttggtc aac gaa gtt acc gaa ttc gct aag act tgt gtt gct gac  151 152 153 154155 156 157 158 159 160 161 162 163 164 165  E   S   A   E   N   C   D   K   S   L   H   T   L   F   G 1279  gaatct gct gaa aac tgt gac aag tcc ttg cac acc ttg ttc ggt  166 167 168 169170 171 172 173 174 175 176 177 178 179 180  D   K   L   C   T   V   A   T   L   R   E   T   Y   G   E 1324  gataag ttg tgt act gtt gct acc ttg aga gaa acc tac ggt gaa  181 182 183 184185 186 187 188 189 190 191 192 193 194 195  M   A   D   C   C   A   K   Q   E   P   E   R   N   E   C 1369  atggct gac tgt tgt gct aag caa gaa cca gaa aga aac gaa tgt  196 197 198 199200 201 202 203 204 205 206 207 208 209 210  F   L   Q   H   K   D   D   N   P   N   L   P   R   L   V 1414  ttcttg caa cac aag gac gac aac cca aac ttg cca aga ttg gtt  211 212 213 214215 216 217 218 219 220 221 222 223 224 225  R   P   E   V   D   V   M   C   T   A   F   H   D   N   S 1459  agacca gaa gtt gac gte atg tgt act get ttc cae gac aae gaa  226 227 228 229230 231 232 233 234 235 236 237 238 239 240  E   T   F   L   K   K   Y   L   Y   E   I   A   R   R   H 1504  gaaacc ttc ttg aag aAG TAC Ttg tac gaa att gct aga aga cac                     ScaI....  241 242 243 244 245 246 247 248 249 250251 252 253 254 255  P   Y   F   Y   A   P   E   L   L   F   F   A   K   R   Y 1549  ccatac ttc tac gct cca gaa ttg ttg ttc ttc gct aag aga tac  256 257 258 259260 261 262 263 264 265 266 267 268 269 270  K   A   A   F   T   E   C   C   Q   A   A   D   K   A   A 1594  aaggct gct ttc acc gaa tgt tgt caa gct gct gat aag gct gct  271 272 273 274275 276 277 278 279 280 281 282 283 284 285  C   L   L   P   K   L   D   E   L   R   D   E   G   K   A 1639  tgtttg ttg cca aag ttg gat gaa ttg aga gac gaa ggt aag gct  286 287 288 289290 291 292 293 294 295 296 297 298 299 300  S   S   A   K   Q   R   L   K   C   A   S   L   Q   K   F 1684  tcttcc gct aag caa aga ttg aag tgt gct tcc ttg caa aag ttc  301 302 303 304305 306 307 308 309 310 311 312 313 314 315  G   E   R   A   F   K   A   W   A   V   A   R   L   S   Q 1729  ggtgaa aga gct ttc aag gct tgg gct gtc gct aga ttg tct caa  316 317 318 319320 321 322 323 324 325 326 327 328 329 330  R   F   P   K   A   E   F   A   E   V   S   K   L   V   T 1774  agattc cca aag gct gaa ttc gct gaa gtt tct aag ttg gtt act  331 332 333 334335 336 337 338 339 340 341 342 343 344 345  D   L   T   K   V   H   T   E   C   C   H   G   D   L   L 1819  gacttg act aag gtt cac act gaa tgt tgt cac ggt gac ttg ttg  346 347 348 349350 351 352 353 354 355 356 357 358 359 360  E   C   A   D   D   R   A   D   L   A   K   Y   I   C   E 1864  gaatgt gct gat gac aga gct gac ttg gct aag tac atc tgt gaa  361 362 363 364365 366 367 368 369 370 371 372 373 374 375  N   Q   D   S   I   S   S   K   L   K   E   C   C   E   K 1909  aaccaa gac tct atC TCT TCc aag ttg aag gaa tgt tgt gaa aag                   EarI....  376 377 378 379 380 381 382 383 384 385 386387 388 389 390  P   L   L   E   K   S   H   C   I   A   E   V   E   N   D 1954  ccattg ttg gaa aag tct cac tgt att gct gaa gtt gaa aac gat  391 392 393 394395 396 397 398 399 400 401 402 403 404 405  E   M   P   A   D   L   P   S   L   A   A   D   F   V   E 1999  gaaatg cCA GCT Gac ttg cca tct ttg gct gct gac ttc gtt gaa          PvuII...  406 407 408 409 410 411 412 413 414 415 416 417 418419 420   S   K   D   V   C   K   N   Y   A   E   A   K   D   V   F 2044 tct aag gac gtt tgt aag aac tac gct gaa gct aag gac gtc ttc  421 422423 424 425 426 427 428 429 430 431 432 433 434 435  L   G   M   F   L   Y   E   Y   A   R   R   H   P   D   Y 2089  ttgggt atg ttc ttg tac gaa tac gct aga aga cac cca gac tac  436 437 438 439440 441 442 443 444 445 446 447 448 449 450  S   V   V   L   L   L   R   L   A   K   T   Y   E   T   T 2134  tccgtt gtc ttg ttg tig aga ttg gct aag acc tac gaa act acc  451 452 453 454455 456 457 458 459 460 461 462 463 464 465  L   E   K   C   C   A   A   A   D   P   H   E   C   Y   A 2179  ttggaa aag tgt tgt gct gct gct gac cca cac gaa tgt tac gct  466 467 468 469470 471 472 473 474 475 476 477 478 479 480  K   V   F   D   E   F   K   P   L   V   E   E   P   Q   N 2224  aaggtt ttc gat gaa ttc aag cca ttg gtc gaa gaa cca caa aac  481 482 483 484485 486 487 488 489 490 491 492 493 494 495  L   I   K   Q   N   C   E   L   F   E   Q   L   G   E   Y 2269  tTGATC Aag caa aac tgt gaa ttg ttc gaa caa ttg ggt gaa tac   BclI....  496497 498 499 500 501 502 503 504 505 506 507 508 509 510  K   F   Q   N   A   L   L   V   R   Y   T   K   K   V   P 2314  aagttc caa aac gct ttg ttg gtt aga tac act aag aag gtc cca  511 512 513 514515 516 517 518 519 520 521 522 523 524 525  Q   V   S   T   P   T   L   V   E   V   S   R   N   L   G 2359  caagtc tCC Acc cca act tTG Gtt gaa gtc TCT AGA aac ttg ggt          XcmI................           XbaI...(2/2)  526 527 528 529530 531 532 533 534 535 536 537 538 539 540  K   V   G   S   K   C   C   K   H   P   E   A   K   R   M 2404  aaggtc ggt tct aag tgt tgt aag cac cca gaa gct aag aGA ATG                                                       BsmI....  541 542543 544 545 546 547 548 549 550 551 552 553 554 555  P   C   A   E   D   Y   L   S   V   V   L   N   Q   L   C 2449 Cca tgtgct gaa gat tac ttg tcc gtc gtt ttg aac caa ttg tgt BsmI..  556 557 558559 560 561 562 563 564 565 566 567 568 569 570  V   L   H   E   K   T   P   V   S   D   R   V   T   K   C 2494  gttttg cac gaa aaG ACc cca GTC tct gat aga gtC ACc aaG TGt                   PshAI........               DraIII......                          AlwNI  571 572 573 574 575 576 577 578 579 580581 582 583 584 585  C   T   E   S   L   V   N   R   R   P   C   F   S   A   L 2539  tgtact gaa tct ttg GTT AAC aga aga cca tgt ttc tct gct ttg                     HpaI...  586 587 588 589 590 591 592 593 594 595596 597 598 599 600  E   V   D   E   T   Y   V   P   K   E   F   N   A   E   T 2584  gaaGTC GAC gaa act tac gtt cca aag gaa ttc aac gct gaa act      SalI... 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615  F   T   F   H   A   D   I   C   T   L   S   E   K   E   R 2629  ttcacc ttc cac gct GAT ATC tgt acc ttg tcc gaa aag gaa aga                     EcoRV..  616 617 618 619 620 621 622 623 624 625626 527 628 629 630  Q   I   K   K   Q   T   A   L   V   E   L   V   K   H   K 2674  caaatt aag aag caa act gct ttg gtt gaa ttg gtc aag cac aag  631 632 633 634635 636 637 638 639 640 641 642 643 644 645  P   K   A   T   K   E   Q   L   K   A   V   M   D   D   F 2719  ccaaag gct act aag gaa caa ttg aag gct gtc atg gat gat ttc  646 647 648 649650 651 652 653 654 655 656 657 658 659 660  A   A   F   V   E   K   C   C   K   A   D   D   K   E   T 2764  gctgct ttc gtt gaa aag tgt tgt aag gat gat gat aag gaa act  661 662 663 664665 666 667 668 669 670 671 672 673 674 675  C   F   A   E   S   G   K   K   L   V   A   A   S   Q   A 2809  tgtttc gct gaa gaa ggt aag aag ttg gtc got gct tcc caa gct  676 677 678 679680 681 682 683 684 685 686 687 688 689 690  A   L   G   L   G   G   S   G   G   S   G   G   S   G   G 2854  gCCTTA GGc tta ggt ggt tct ggt ggt tcc ggt ggt TCC GGA ggt  Bsu36I...                                      BspEI..  691 692 693694   S   G   G   T 2899  tcc ggt GGT ACC     taa tAA GCTTa attcttatga         KpnI...     Stop Stop                        HindIII (2/2) 2932 tttatgattt ttattattaa ataagTTATA Aaaaaaataa gtGTATACaa attttaaagt                            PsiI...            BstZ17I 2992  gactcttaggttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg 3052  ttgctttctcaggtatagca tgaggtcgct cttattgacc acacctctac cgGCATGCcg                                                          SphI.. 3112 agcaaatgcc tgcaaatcgc tccccatttc acccaattgt agatatgcta actccagcca 3172 tgagttgatg aatctcggtg tgtattttat gtcctcagag gacaacacct gttgtaatcg 3232 ttcttccaca cggatCGCGG CCGC                  NotI

TABLE 27 DNA to insert at BspEI/KpnI site for 2^(nd) encoding of DX-890(SEQ. ID NO:_(——)) TCCGGAggta gtggtggctc cggtggtgag gcttgcaatCttcctatcgt Ccgtggccct tgcatcgcct tttttcctcg ttgggccttt gacgccgtcaAaggcaaatg cgtccttttt ccttacggcg gttgccaggg caatggcaat Aaattttatagcgagaaaga gtgccgtgag tattgcggcg tcccttaata aGGTACC

TABLE 28 NotI cassette of pDB2300X3 with 2 x DX890 DNA sequence has SEQID NO: _(——) AA Sequence has SEQ ID NO: Enzymes that cut from 1 to 3times. $ =DAM site, * = DCM site, & = both NotI GCggccgc 2 1 3434 EagICggccg 2 2 3435 KasI Ggcgcc 1 160 AfeI AGCgct 1 193 NaeI GCCggc 1 234NgoMIV Gccggc 1 234 BsgI ctgcac 1 450 BcgI gcannnnnntcg 1 568 BanIIGRGCYc 1 620 PatI CTGCAg 1 636 AflIl Cttaag 1 763 HindIII Aagctt 2 8013101 BglII Agatct 1 883$ PflMI CCANNNNntgg 1 994 Ndel CAtatg 1 995 BamHIGgatcc 1 1072$ AgeI Accggt 1 1136 AvrII Cctagg 1 1149 BmgBI CACgtc 11225$ ScaI AGTact 1 1520 EarI CTCTTCNnnn 1 1923 PvuII CAGctg 1 2006 BclITgatca 1 2270$ XcmI CCANNNNNnnnntgg 1 2366 BamI GAATGCN 1 2444 PshAIGACNNnngtc 1 2508 AlwNI CAGNNNctg 1 2513 DraIII CACNNNgtg 1 2529 HpaIGTTaac 1 2554 SalI Gtcgac 1 2587 ECoRV GATatc 1 2644 Bsu36I CCtnagg 12855 BspEI Tccgga 1 2890 PflFI GACNnngtc 1 2980 Tth111I GACNnngtc 1 2980Acc65I Ggtacc 1 3091 KpnI GGTACc 1 3091 PsiI TTAtaa 1 3143 BstZ17IGTAtac 1 3160 SphI GCATGc 1 3290-------------------------------------------- (SEQ. ID NO:_(——)). 1GCGGCCGCcc gtaatgcggt atcgtgaaag cgaaaaaaaa actaacagta gataagacagNotI.... 61 atagacagat agagatggac gagaaacagg gggggagaaa aggggaaaagagaaggaaag 121 aaagactcat ctatcgcaga taagacaatc aaccctcatG GCGCCtccaaccaccatccg                                           NarI... 181cactagggac caAGCGCTcg caccgttagc aacgcttgac tcacaaacca actGCCGGCt         AfeI..                                       NgoMIV 241gaaagagctt gtgcaatggg agtgccaatt caaaggagcc gaatacgtct gctcgccttt 301taagaggctt tttgaacact gcattgcacc cgacaaatca gccactaact acgaggtcac 361ggacacatat accaatagtt aaaaattaca tatactctat atagcacagt agtgtgataa 421ataaaaaatt ttgccaagac ttttttaaaC TGCACccgac agatcaggtc tgtgcctact                               BsgI... 481 atgcacttat gcccggggtcccgggaggag aaaaaacgag ggctgggaaa tgtccgtgga 541 ctttaaacgc tccgggttagcagagtaGCA gggcttTCGg ctttggaaat ttaggtgact                             BCgI 601 tgttgaaaaa gcaaaatttg ggctcagtaatgCCActgca gTGGcttatc acgccaggac                                   BstXI........                                      PStI... 661 tgcgggagtg gcgggggcaaacacacccgc gataaagagc gcgatgaata taaaaggggg 721 ccaatgttac gtcccgttatattggagttc ttcccataca aaCTTAAGag tccaattagc                                              AflII. 781 ttcatcgccaataaaaaaac AAGCTTaacc taattctaac aagcaaag                       HindIII(1/2) Signal sequence------------------------------------------>  1   2   3   4   5   6   7   8   9   10  11  12  13  14  15 M   K   W   V   F   I   V   S   I   L   F   L   F   S   S 829 atg aagtgg gtt ttc atc gtc tcc att ttg ttc ttg ttc tcc tct Signalsequence------------------> DXB90, first instance --> 16  17  18  19  20  21  22  23  24  25  26  27  28  29  30 A   Y   S   R   S   L   D   K   R   E   A   C   N   L   P 874 gct tactct AGA TCT ttg gat aag aga gaa gcc tgt aac ttg cca             BglII.. 31  32  33  34  35  36  37  38  39  40  41  42  43  44  45 I   V   R   G   P   C   I   A   F   F   P   R   W   A   F 919 att gttaga ggt cca tgt att gct ttc ttc cca aga tgg gct ttc 46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 D   A   V   K   G   K   C   V   L   F   P   Y   G   G   C 964 gat gctgtt aag ggt aag tgt gtt ttg ttc CCA tat ggT GGt tgt                                        PflMI                                         NdeI. 61  62  63  64  65  66  67  68  69  70  71  72  73  74  75 Q   G   N   G   N   K   F   Y   S   E   K   E   C   R   E 1009 caa ggtaac ggt aac aag ttc tac tct gaa aag gaa tgt aga gaa----DX890#1------>--------------- Linker ---------------- 76  77  78  79  80  81  82  83  84  85  86  87  88  89  90 Y   C   G   V   P   G   G   S   G   G   S   G   G   S   G 1054 tac tgtggt gtt cca ggt GGA TCC ggt ggt tcc ggt ggt tct ggt                        BamHI.. --- Linker --->------------- rHA gene----until codon 679 -->  91  92  93  94  95  96  97  98  99 100 101 102103 104 105  G   S   G   G   D   A   H   K   S   E   V   A   H   R   F1099  ggt tcc ggt ggt gac gct cac aag tcc gaa gtc gct cAC CGG Ttc                                                 AgeI....  106 107 108109 110 111 112 113 114 115 116 117 118 119 120 K   D   L   G   E   E   N   F   K   A   L   V   L   I   A 1144  aag gaCCTA GGt gag gaa aac ttc aag gct ttg gtc ttg atc gct       AvrII...  121122 123 124 125 126 127 128 129 130 131 132 133 134 135 F   A   Q   Y   L   Q   Q   C   P   F   E   D   H   V   K 1189  ttc gctcaa tac ttg caa caa tgt cca ttc gaa gat cac gtc aag  136 137 138 139 140141 142 143 144 145 146 147 148 149 150 L   V   N   B   V   T   E   F   A   K   T   C   V   A   D 1234  ttg gtcaac gaa gtt acc gaa ttc gct aag act tgt gtt gct gac  151 152 153 154 155156 157 158 159 160 161 162 163 164 165 E   S   A   E   N   C   D   K   S   L   H   T   L   F   G 1279  gaa tctgct gaa aac tgt gac aag tcc ttg cac acc ttg ttc ggt  166 167 168 169 170171 172 173 174 175 176 177 178 179 180 D   K   L   C   T   V   A   T   L   R   E   T   Y   G   E 1324  gat aagttg tgt act gtt gct acc ttg aga gaa acc tac ggt gaa  181 182 183 184 185186 187 188 189 190 191 192 193 194 195 M   A   D   C   C   A   K   Q   E   P   E   R   N   E   C 1369  atg gctgac tgt tgt gct aag caa gaa cca gaa aga aac gaa tgt  196 197 198 199 200201 202 203 204 205 206 207 208 209 210 F   L   Q   H   K   D   D   N   P   N   L   P   R   L   V 1414  ttc ttgcaa cac aag gac gac aac cca aac ttg cca aga ttg gtt  211 212 213 214 215216 217 218 219 220 221 222 223 224 225 R   P   E   V   D   V   M   C   T   A   F   H   D   N   E 1459  aga ccagaa gtt gac gtc atg tgt act gct ttc cac gac aac gaa  226 227 228 229 230231 232 233 234 235 236 237 238 239 240 E   T   F   L   K   K   Y   L   Y   E   I   A   R   R   H 1504  gaa accttc ttg aag aag tac ttg tac gaa att gct aga aga cac  241 242 243 244 245246 247 248 249 250 251 252 253 254 255 P   Y   F   Y   A   P   E   L   L   F   F   A   K   R   Y 1549  cca tacttc tac gct cca gaa ttg ttg ttc ttc gct aag aga tac  256 257 258 259 260261 262 263 264 265 266 267 268 269 270 K   A   A   F   T   E   C   C   Q   A   A   D   K   A   A 1594  aag gctgct ttc acc gaa tgt tgt caa gct gct gat aag gct gct  271 272 273 274 275276 277 278 279 280 281 282 283 284 285 C   L   L   P   K   L   D   E   L   R   D   E   G   K   A 1639  tgt ttgttg cca aag ttg gat gaa ttg aga gac gaa ggt aag gct  286 287 288 289 290291 292 293 294 295 296 297 298 299 300 S   S   A   K   Q   R   L   K   C   A   S   L   Q   K   F 1684  tct tccgct aag caa aga ttg aag tgt gct tcc ttg caa aag ttc  301 302 303 304 305306 307 308 309 310 311 312 313 314 315 G   E   R   A   F   K   A   W   A   V   A   R   L   S   Q 1729  ggt gaaaga gct ttc aag gct tgg gct gtc gct aga ttg tct caa  316 317 318 319 320321 322 323 324 325 326 327 328 329 330 R   F   P   K   A   E   F   A   E   V   S   K   L   V   T 1774  aga ttccca aag gct gaa ttc gct gaa gtt tct aag ttg gtt act  331 332 333 334 335336 337 338 339 340 341 342 343 344 345 D   L   T   K   V   H   T   E   C   C   H   G   D   L   L 1819  gac ttgact aag gtt cac act gaa tgt tgt cac ggt gac ttg ttg  346 347 348 349 350351 352 353 354 355 356 357 358 359 360 E   C   A   D   D   R   A   D   L   A   K   Y   I   C   E 1864  gaa tgtgct gat gac aga gct gac ttg gct aag tac atc tgt gaa  361 362 363 364 365366 367 368 369 370 371 372 373 374 375 N   Q   D   S   I   S   S   K   L   K   E   C   C   E   K 1909  aac caagac tct atc tct tcc aag ttg aag gaa tgt tgt gaa aag  376 377 378 379 380381 382 383 384 385 386 387 388 389 390 P   L   L   E   K   S   H   C   I   A   E   V   E   N   D 1954  cca ttgttg gaa aag tct cac tgt att gct gaa gtt gaa aac gat  391 392 393 394 395396 397 398 399 400 401 402 403 404 405 E   M   P   A   D   L   P   S   L   A   A   D   F   V   E 1999  gaa atgcca gct gac ttg cca tct ttg gct gct gac ttc gtt gaa  406 407 408 409 410411 412 413 414 415 416 417 418 419 420 S   K   D   V   C   K   N   Y   A   E   A   K   D   V   F 2044  tct aaggac gtt tgt aag aac tac gct gaa gct aag gac gtc ttc  421 422 423 424 425426 427 428 429 430 431 432 433 434 435 L   G   M   F   L   Y   S   Y   A   R   R   H   P   D   Y 2089  ttg ggtatg ttc ttg tac gaa tac gct aga aga cac cca gac tac  436 437 438 439 440441 442 443 444 445 446 447 448 449 450 S   V   V   L   L   L   R   L   A   K   T   Y   S   T   T 2134  tcc gttgtc ttg ttg ttg aga ttg gct aag acc tac gaa act acc  451 452 453 454 455456 457 458 459 460 461 462 463 464 465 L   E   K   C   C   A   A   A   D   P   H   E   C   Y   A 2179  ttg gaaaag tgt tgt gct gct gct gac cca cac gaa tgt tac gct  466 467 468 469 470471 472 473 474 475 476 477 478 479 480 K   V   F   D   S   F   K   P   L   V   E   E   P   Q   N 2224  aag gttttc gat gaa ttc aag cca ttg gtc gaa gaa cca caa aac  481 482 483 484 485486 487 488 489 490 491 492 493 494 495 L   I   K   Q   N   C   E   L   F   E   Q   L   G   E   Y 2269  ttg atcaag caa aac tgt gaa ttg ttc gaa caa ttg ggt gaa tac  496 497 498 499 500501 502 503 504 505 506 507 508 509 510 K   F   Q   N   A   L   L   V   R   Y   T   K   K   V   P 2314  aag ttccaa aac gct ttg ttg gtt aga tac act aag aag gtc cca  511 512 513 514 515516 517 518 519 520 521 522 523 524 525 Q   V   S   T   P   T   L   V   E   V   S   R   N   L   G 2359  caa gtctcc acc cca act ttg gtt gaa gtc tct aga aac ttg ggt  526 527 528 529 530531 532 533 534 535 536 537 538 539 540 K   V   G   S   K   C   C   K   H   P   E   A   K   R   M 2404  aag gtcggt tct aag tgt tgt aag cac cca gaa gct aag aga atg  541 542 543 544 545546 547 548 549 550 551 552 553 554 555 P   C   A   E   D   Y   L   S   V   V   L   N   Q   L   C 2449  cca tgtgct gaa gat tac ttg tcc gtc gtt ttg aac caa ttg tgt  556 557 558 559 560561 562 563 564 565 566 567 568 569 570 V   L   H   S   K   T   P   V   S   D   R   V   T   K   C 2494  gtt ttgcac gaa aag acc cca gtc tct gat aga gtc acc aag tgt  571 572 573 574 575576 577 578 579 580 581 582 583 584 585 C   T   E   S   L   V   N   R   R   P   C   F   S   A   L 2539  tgt actgaa tct ttg gtt aac aga aga cca tgt ttc tct gct ttg  586 587 588 589 590591 592 593 594 595 596 597 598 599 600 S   V   D   S   T   Y   V   P   K   S   F   N   A   E   T 2584  gaa gtcgac gaa act tac gtt cca aag gaa ttc aac gct gaa act  601 602 603 604 605606 607 608 609 610 611 612 613 614 615 F   T   F   H   A   D   I   C   T   L   S   S   K   E   R 2629  ttc accttc cac gct gat atc tgt acc ttg tcc gaa aag gaa aga  616 617 618 619 620621 622 623 624 625 626 627 628 629 630 Q   I   K   K   Q   T   A   L   V   S   L   V   K   H   K 2674  caa attaag aag caa act gct ttg gtt gaa ttg gtc aag cac aag  631 632 633 634 635636 637 638 639 640 641 642 643 644 645 P   K   A   T   K   E   Q   L   K   A   V   M   D   D   F 2719  cca aaggct act aag gaa caa ttg aag gct gtc atg gat gat ttc  646 647 648 649 650651 652 653 654 655 656 657 658 659 660 A   A   F   V   E   K   C   C   K   A   D   D   K   E   T 2764  gct gctttc gtt gaa aag tgt tgt aag gct gat gat aag gaa act  661 662 663 664 665666 667 668 669 670 671 672 673 674 675 C   F   A   E   E   G   K   K   L   V   A   A   S   Q   A 2809  tgt ttcgct gaa gaa ggt aag aag ttg gtc gct gct tcc caa gct                Linker--------------------------------->  676 677 678679680 681 682 683 684 685 686 687 688 689 690 A   L   G   L   G   G   S   G   G   S   G   G   S   G   G 2854  gCC TTAGGc tta ggt ggt tct ggt ggt tcc ggt ggt TCC GGA ggt Bsu36I...                                      BspEI..                        DX-890(seccnd encoding)----to end-->>  691 692693 694 695 696 697 698 699 700 701 702 703 704 705 S   G   G   S   G   G   E   A   C   N   L   P   I   V   R 2899  agt ggtggc tcc ggt ggt gag gct tgc aat ctt cct atc gtc cgt  706 707 708 709 710711 712 713 714 715 716 717 718 719 720 G   P   C   I   A   F   F   P   R   W   A   F   D   A   V 2944  ggc ccttgc atc gcc ttt ttt cct cgt tgg gcc ttt gac gcc gtc  721 722 723 724 725726 727 728 729 730 731 732 733 734 735 K   G   K   C   V   L   F   P   Y   G   G   C   Q   G   N 2989  aaa ggcaaa tgc gtc ctt ttt cct tac ggc ggt tgc cag ggc aat  736 737 738 739 740741 742 743 744 745 746 747 748 749 750 G   N   K   F   Y   S   E   K   E   C   R   E   Y   C   G 3034  ggc aataaa ttt tat agc gag aaa gag tgc cgt gag tat tgc ggc  751 752  V   P 3079 gtc cct taa taa        GGT ACC           taa  tAA GCTTa attcttatga                         KpnI...          Stop Stop                                      HindIII (2/2) 3118 tttatgatttttattattaa ataagTTATA Aaaaaaataa gtGTATACaa attttaaagt                      PsiI...            BstZ17I 3178 gactcttaggttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg 3238 ttgctttctcaggtatagca tgaggtcgct cttattgacc acacctctac cgGCATGCcg                                                                                                                         SphI..3298 agcaaatgcc tgcaaatcgc tccccatttc acccaattgt agatatgcta actccagcaa3358 tgagttgatg aatctcggtg tgtattttat gtcctcagag gacaacacct gttgtaatcg3418 ttcttccaca cggatCGCGG CCGC                  NotI

TABLE 29 AA sequence of DX890::(GGS)4GG::HA::(GGS)4GG::DX890 (SEQ IDNO:_) EACNLPIVRG PCIAFFPRWA FDAVKGKCVL FPYGGCQGNG NKFYSEKECR EYCGVPGGSGGSGGSGGSGG DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFAKTCVADESAE NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNPNLPRLVRPEV DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQAADKAACLLP KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAEVSKLVTDLTK VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCIAEVENDEMPA DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLAKTYETTLEKC CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVRYTKKVPQVST PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE DYLSVVLNQL CVLHEKTPVSDRVTKCCTES LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALVELVKHKPKAT KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGLGGSGGSGGSGGSGGS GGEACNLPIV RGPCIAFFPR WAFDAVKGKC VLFPYGGCQG NGNKFYSEKECREYCGVP

TABLE 30 DNA sequence of the N-terminal BglII-BamHI DX-1000 cDNA (SEQ IDNO:_(——)) AGA TCT TTG GAT AAG AGA gag gct atg cat tcc ttc tgc gcc ttcaag gct gag act ggt cct tgt aga gct agg ttc gac cgt tgg ttc ttc aac atcttc acg cgt cag tgc gag gaa ttc att tac ggt ggt tgt gaa ggt aac cag aaccgg ttc gaa tct cta gag gaa tgt aag aag atg tgc act cgt gac GGA TCC

TABLE 31 AA sequence of DX1000::(GGS)4GG::HA (SEQ ID NO:_(——) EAMHSFCAFKAETGPCRARF DRWFFNIFTR QCEEFIYGGC EGNQNRFESL EECKKMCTRD GGSGGSGGSGGSGGDAHKSE VAHRFKDLGE ENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVADESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEP ERNECFLQHK DDNPNLPRLVRPEVDVMCTA FHDNEETFLK KYLYEIARRM PYFYAPELLF FAKRYKAAFT ECCQAADKAACLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAV ARLSQRFPKA EFAEVSKLVTDLTKVHTECC HGDLLECADD RADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVENDEMPADLPSLA ADFVESKDVC KMYAEAKDVF LGMFLYEYAR RHPDYSVVLL LRLAKTYETTLEKCCAAADP HECYAKVFDE FKPLVEEPQN LIKQNCELFE QLGEYKFQNA LLVRYTKKVPQVSTPTLVEV SRNLGKVGSK CCKHPEAKRM PCAEDYLSVV LNQLCVLHEK TPVSDRVTKCCTESLVNRRP CFSALEVDET YVPKEFNAET FTFHADICTL SEKERQIKKQ TALVELVKHKPKATKEH

TABLE 32 DNA sequence of the N-terminal BspEI-KpnI DX-88cDNA-2^(nd) encoding (SEQ ID NO:_(——)) TCC GGA ggt agt ggt ggc tcc ggtggt GAg GCc ATG CAt TCT TTC TGT GCT TTC AAG GCT GAC GAC GGT CCG TGC AGAGCT GCT CAC CCA AGA TGG TTC TTC AAC ATC TTC ACG CGA CAA TGC GAG GAG TTCATC TAC GGT GGT TGT GAG GGT AAC CAA AAC AGA TTC GAG TCT CTA GAG GAG TGTAAG AAG ATG TGT ACT AGA GAC GGT taa taa GGT ACC

TABLE 33 AA sequence of DPI14::HSA (SEQ ID NO:_(——)) EAVREVCSEQAETGPCIAFF PRWYFDVTEG KCAPFFYGGC GGNRNNFDTE EYCMAVCGSA GGSGGSGGSGGSGGDAHKSE VAHRFKDLGE ENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVADESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEP ERNECFLQHK DDNPNLPRLVRPEVDVMCTA FHDNEETFLK KYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAACLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAV ARLSQRFPKA EFAEVSKLVTDLTKVHTECC HGDLLECADD RADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVENDEMPADLPSLA ADFVESKDVC KNYAEAKDVF LGMFLYEYAR RHPDYSVVLL LRLAKTYETTLEKCCAAADP HECYAKVFDE FKPLVEEPQN LIKQNCELFE QLGEYKFQNA LLVRYTKKVPQVSTPTLVEV SRNLGKVGSK CCKHPEAKRM PCAEDYLSVV LNQLCVLHEK TPVSDRVTKCCTESLVNRRP CFSALEVDET YVPKEFNAET FTFHADICTL SEKERQIKKQ TALVELVKHKPKATKEH

1. An albumin fusion protein comprising a Kunitz domain peptide or afragment or variant thereof, and albumin, or a fragment or variantthereof.
 2. The albumin fusion protein according to claim 1, wherein theKunitz domain peptide or a fragment or variant thereof has a functionalactivity.
 3. The albumin fusion protein according to claim 2, whereinthe functional activity comprises inhibiting serine proteases.
 4. Thealbumin fusion protein according to claim 2, wherein the functionalactivity comprises inhibiting plasmin.
 5. The albumin fusion proteinaccording to claim 2, wherein the functional activity comprisesinhibiting human neutrophil elastase.
 6. The albumin fusion proteinaccording to claim 2, wherein the functional activity comprisesinhibiting kallikrein.
 7. The albumin fusion protein according to claim1 comprising DX-890 or a fragment or variant thereof and albumin or afragment or variant thereof.
 8. The albumin fusion protein according toclaim 1 comprising DPI-14 or a fragment or variant thereof and albuminor a fragment or variant thereof.
 9. The albumin fusion proteinaccording to claim 1 comprising DX-88 or a fragment or variant thereofand albumin or a fragment or variant thereof.
 10. The albumin fusionprotein according to claim 1 comprising DX-1000 or a fragment or variantthereof and albumin or a fragment or variant thereof.
 11. The albuminfusion protein according to claim 1 wherein the albumin fusion proteincomprises at least two Kunitz domain fusion peptides or fragments orvariants thereof.
 12. The albumin fusion protein according to claim 11,wherein each of the at least two Kunitz domain fusion peptides orfragments or variants thereof has a functional activity.
 13. The albuminfusion protein according to claim 12, wherein the functional activity ofone of the at least two Kunitz domain fusion peptides comprisesinhibiting serine proteases.
 14. The albumin fusion protein according toclaim 12, wherein the functional activity of one of the at least twoKunitz domain fusion peptides comprises inhibiting plasmin.
 15. Thealbumin fusion protein according to claim 12, wherein the functionalactivity of one of the at least two Kunitz domain fusion peptidescomprises inhibiting human neutrophil elastase.
 16. The albumin fusionprotein according to claim 12, wherein the functional activity of one ofthe at least two Kunitz domain fusion peptides comprises inhibitingkallikrein.
 17. The albumin fusion protein according to claim 11 whereinat least two of the Kunitz domain peptides or fragments or variantsthereof have different amino acid sequences.
 18. The albumin fusionprotein of claim 1 comprising at least one fragment or variant of apeptide selected from the group consisting of DX-890, DX-88, DX-1000,and DPI-14 and albumin or a fragment or variant thereof, and whereinsaid albumin fragment or variant has albumin activity and said peptidefragment or variant has a functional activity.
 19. The albumin fusionprotein according to claim 1, wherein said albumin activity has theability to prolong the in vivo half-life of a peptide selected from thegroup consisting of DX-890, DX-88, DX-1000, and DPI-14, or a fragment orvariant thereof, compared to the in vivo half-life of the peptide or afragment or variant thereof in an unfused state.
 20. The albumin fusionprotein according to claim 1, further comprising or one or moreadditional albumin moieties.
 21. The albumin fusion protein according toclaim 1, wherein the albumin fusion protein comprises one or moremoieties selected from the group consisting of DX-890, DX-88, DX-1000,and DPI-14, or fragments or variants thereof, or one or more additionalalbumin moieties.
 22. The albumin fusion protein according to claim 1,wherein said fusion protein further comprises a chemical moiety.
 23. Thealbumin fusion protein according to claim 1, wherein the Kunitz domainpeptide, or fragment or variant thereof, is fused to the N-terminus ofalbumin or to the N-terminus of the fragment or variant of albumin. 24.The albumin fusion protein according to claim 23, wherein the Kunitzdomain peptide comprises DX-890, DPI-14, DX-88, or DX-1000.
 25. Thealbumin fusion protein of claim 1, wherein the Kunitz domain peptide orfragment of variant thereof, is fused to the C-terminus of albumin, orthe C-terminus of the fragment or variant of albumin.
 26. The albuminfusion protein according to claim 24, wherein the Kunitz domain peptidecomprises DX-890, DPI-14, DX-88, or DX-1000.
 27. The albumin fusionprotein according to claim 1, wherein said Kunitz domain peptidecomprises a first peptide, or fragment or variant thereof, and a secondpeptide, or fragment or variant thereof, and wherein said peptide, orfragment or variant thereof, is different from said second peptide, orfragment or variant thereof.
 28. The albumin fusion protein according toclaim 27, wherein said first peptide, or fragment or variant thereof,and said second peptide, or fragment or variant thereof is chosen fromthe group consisting of DX-890, DX-88, DX-1000, and DPI-14.
 29. Thealbumin fusion protein according to claim 1, wherein the Kunitz domainpeptide, or fragment or variant thereof, is separated from the albuminor the fragment or variant of albumin by a linker.
 30. The albuminfusion protein according to claim 1, wherein the albumin fusion proteincomprises the following formula: R2-R1; R1-R2; R2-R1-R2; R2-L-R1-L-R2;R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, wherein R1 is at least one peptideselected from the group consisting of DX-890, DX-88, DX-1000, andDPI-14, or a fragment or variant thereof, Lisa peptide linker, and R2 isalbumin.
 31. The albumin fusion protein according to claim 1, whereinthe in vitro biological activity of the Kunitz domain peptide, orfragment or variant thereof, fused to albumin, or fragment or variantthereof, is greater than the in vitro biological activity of the Kunitzdomain peptide, or fragment or variant thereof, in an unfused state. 32.The albumin fusion protein according to claim 1, wherein the solubilityof the Kunitz domain peptide, or fragment or variant thereof, fused toalbumin, or fragment or variant thereof, is greater than the solubilityof the Kunitz domain peptide, or fragment or variant thereof, in anunfused state that has been subjected to the same storage, handling orphysiological conditions.
 33. The albumin fusion protein according toclaim 30, wherein the in vivo biological activity of the at least onepeptide, or fragment or variant thereof, fused to albumin, or fragmentor variant thereof, is greater than the in vivo biological activity ofthe at least one peptide, or fragment or variant thereof, in an unfusedstate.
 34. The albumin fusion protein according to claim 1, wherein thealbumin fusion protein is non-glycosylated.
 35. The albumin fusionprotein according to claim 1, wherein the albumin fusion protein isexpressed in yeast.
 36. The albumin fusion protein according to claim35, wherein the yeast is glycosylation deficient.
 37. The albumin fusionprotein according to claim 36 wherein the yeast is protease deficient.38. The albumin fusion protein according to claim 1, wherein the albuminfusion protein is expressed by a mammalian cell.
 39. The albumin fusionprotein according to claim 38, wherein the albumin fusion protein isexpressed by a mammalian cell in culture.
 40. A composition comprisingthe albumin fusion protein of claim 1 and a pharmaceutically acceptablecarrier.
 41. A method of treating a disease or disorder in a patient,comprising the step of administering the albumin fusion protein ofclaim
 1. 42. A method of treating a patient with cystic fibrosis or acystic fibrosis-related disease or disorder that is modulated by DX-890,DPI-14, or DX-890 and DPI-14 comprising the step of administering aneffective amount of the albumin fusion protein of claim 1, wherein saidKunitz domain peptide is DX-890 or DPI-14, or a fragment or variantthereof.
 43. A method of extending the in vivo half-life of DX-890,DPI-14, or DX-890 and DPI-14, or a fragment or variant thereof,comprising the step of fusing the DX-890, and/or DPI-14, or DX-890 andDPI-14, or fragment or variant thereof, to albumin or a fragment orvariant of albumin sufficient to extend the in vivo half-life of theDX-890, DPI-14, or DX-890 and DPI-14, or fragment or variant thereof,compared to the in vivo half-life of the DX-890, DPI-14, or DX-890 andDPI-14, or fragment or variant thereof, in an unfused state.
 44. Amethod of treating a patient with hereditary angioedema or a hereditaryangioedema-related disease or disorder that is modulated by DX-88,comprising the step of administering an effective amount of the albuminfusion protein of claim 1, wherein said Kunitz domain peptide is DX-88,or a fragment or variant thereof.
 45. A method of extending the in vivohalf-life of DX-88, or a fragment or variant thereof, comprising thestep of fusing the DX-88, or fragment or variant thereof, to albumin ora fragment or variant of albumin sufficient to extend the in vivohalf-life of the DX-88, or fragment or variant thereof, compared to thein vivo half-life of the DX-88, or fragment or variant thereof, in anunfused state.
 46. A method of treating a patient with cancer, acancer-related disease, bleeding, or disorder that is modulated byDX-1000, comprising the step of administering an effective amount of thealbumin fusion protein of claim 1, wherein said Kunitz domain peptide isDX-1000, or a fragment or variant thereof.
 47. A method of extending thein vivo half-life of DX-1000, or a fragment or variant thereof,comprising the step of fusing the DX-1000, or fragment or variantthereof, to albumin or a fragment or variant of albumin sufficient toextend the in vivo half-life of the DX-1000, or fragment or variantthereof, compared to the in vivo half-life of the DX-1000 or fragment orvariant thereof, m an unfused state.
 48. A nucleic acid moleculecomprising a polynucleotide sequence encoding the albumin fusion proteinof claim
 1. 49. A vector comprising the nucleic acid molecule of claim48.
 50. A host cell comprising the nucleic acid molecule of claim 48.51. A pharmaceutical composition comprising an effective amount of thealbumin fusion protein of claim 1 and a pharmaceutically acceptablecarrier or excipient.
 52. A method for manufacturing a albumin fusionprotein of claim 1, the method comprising: (a) providing a nucleic acidcomprising a nucleotide sequence encoding the albumin fusion proteinexpressible in an organism; (b) expressing the nucleic acid in theorganism to form an albumin fusion protein; and (c) purifying thealbumin fusion protein.
 53. The method of claim 52 wherein the albuminfusion protein comprises DX-890, DPI-14, or DX-890 and DPI14 albuminfusion expressed in a glycosylation deficient yeast strain.